Transcriptional and post-transcriptional regulation of NK cell development and function

PubMed Central

Leong, Jeffrey W.; Wagner, Julia A.; Ireland, Aaron R.; Fehniger, Todd A.

2016-01-01

Natural killer (NK) cells are specialized innate lymphoid cells that survey against viral infections and malignancy. Numerous advances have improved our understanding of the molecular mechanisms that control NK cell development and function over the past decade. These include both studies on the regulatory effects of transcription factors and translational repression via microRNAs. In this review, we summarize our current knowledge of DNA-binding transcription factors that regulate gene expression and thereby orchestrate NK cell development and activation, with an emphasis on recent discoveries. Additionally, we highlight our understanding of how RNA-bindings microRNAs fine tune the NK cell molecular program. We also underscore the large number of open questions in field that are now being addressed using new technological approaches and genetically engineered model organisms. Ultimately, a deeper understanding of the basic molecular biology of NK cells will facilitate new strategies to manipulate NK cells for the treatment of human disease. PMID:26948928

Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells.

PubMed

Koh, Yung-Hua; Moochhala, Shabbir; Bhatia, Madhav

2012-07-01

Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25-30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10(-6) M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells

PubMed Central

Koh, Yung-Hua; Moochhala, Shabbir; Bhatia, Madhav

2012-01-01

Abstract Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10−6M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R. PMID:22040127

IL-21 augments NK effector functions in chronically HIV-infected individuals

PubMed Central

Strbo, Natasa; de Armas, Lesley; Liu, Huanliang; Kolber, Michael A.; Lichtenheld, Mathias; Pahwa, Savita

2009-01-01

Objective This study addresses the interleukin (IL)-21 effects on resting peripheral blood NK cells in chronically HIV-infected individuals. Design The effects of IL-21 on perforin expression, proliferation, degranulation, IFN-γ production, cytotoxicity and induction of STAT phosphorylation in NK cells were determined in vitro. Methods Peripheral blood mononuclear cells from HIV-infected and healthy individuals were incubated in vitro for 6h, 24h or 5 days with IL-21 or IL-15. Percentages of perforin, IFN-γ, CD107a, NKG2D and STAT3-5 positive cells were determined within NK cell populations. K562 cells were used as target cells in NK cytotoxicity assay. Results Frequency of CD56dim cells in chronically HIV-infected individuals was diminished. Perforin expression in CD56dim and CD56bright was comparable in healthy and HIV-infected individuals. IL-15 up-regulated perforin expression primarily in CD56bright NK cells while IL-21 up-regulated perforin in both NK subsets. IL-21 and IL- 15 up-regulated CD107a and IFN-γ as well as NK cytotoxicity. IL-15 predominantly activated STAT5, while IL-21 activated STAT5 and STAT3. IL-15, but not IL-21 increased NK cell proliferation in uninfected and HIV-infected individuals. Conclusion IL-21 augments NK effector functions in chronically HIV-infected individuals and due to its perforin enhancing properties it has potential for immunotherapy or as a vaccine adjuvant. PMID:18670213

Developmental and Functional Control of Natural Killer Cells by Cytokines

PubMed Central

Wu, Yang; Tian, Zhigang; Wei, Haiming

2017-01-01

Natural killer (NK) cells are effective in combating infections and tumors and as such are tempting for adoptive transfer therapy. However, they are not homogeneous but can be divided into three main subsets, including cytotoxic, tolerant, and regulatory NK cells, with disparate phenotypes and functions in diverse tissues. The development and functions of such NK cells are controlled by various cytokines, such as fms-like tyrosine kinase 3 ligand (FL), kit ligand (KL), interleukin (IL)-3, IL-10, IL-12, IL-18, transforming growth factor-β, and common-γ chain family cytokines, which operate at different stages by regulating distinct signaling pathways. Nevertheless, the specific roles of each cytokine that regulates NK cell development or that shapes different NK cell functions remain unclear. In this review, we attempt to describe the characteristics of each cytokine and the existing protocols to expand NK cells using different combinations of cytokines and feeder cells. A comprehensive understanding of the role of cytokines in NK cell development and function will aid the generation of better efficacy for adoptive NK cell treatment. PMID:28824650

Kinome Analysis of Receptor-Induced Phosphorylation in Human Natural Killer Cells

PubMed Central

König, Sebastian; Nimtz, Manfred; Scheiter, Maxi; Ljunggren, Hans-Gustaf; Bryceson, Yenan T.; Jänsch, Lothar

2012-01-01

Background Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation. Results A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. Conclusions The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses. PMID:22238634

Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma.

PubMed

Laouar, Yasmina; Sutterwala, Fayyaz S; Gorelik, Leonid; Flavell, Richard A

2005-06-01

Interferon-gamma and interleukin 12 produced by the innate arm of the immune system are important regulators of T helper type 1 (T(H)1) cell development, but signals that negatively regulate their expression remain controversial. Here we show that transforming growth factor-beta (TGF-beta) controlled T(H)1 differentiation through the regulation of interferon-gamma produced by natural killer (NK) cells. Blockade of TGF-beta signaling in NK cells caused the accumulation of a large pool of NK cells secreting copious interferon-gamma, responsible for T(H)1 differentiation and protection from leishmania infection. In contrast, blockade of TGF-beta signaling in dendritic cells did not affect dendritic cell homeostasis or interleukin 12 production, thus indicating a previously undescribed demarcation of the function of TGF-beta in NK cells versus dendritic cells.

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. Copyright © 2015 by The American Association of Immunologists, Inc.

Th17/T regulator cell balance and NK cell numbers in relation to psychosis liability and social stress reactivity.

PubMed

Counotte, J; Drexhage, H A; Wijkhuijs, J M; Pot-Kolder, R; Bergink, V; Hoek, H W; Veling, W

2018-03-01

Psychotic disorders are characterized by a deranged immune system, including altered number and function of Natural Killer (NK) and T cells. Psychotic disorders arise from an interaction between genetic vulnerability and exposure to environmental risk factors. Exposure to social adversity during early life is particularly relevant to psychosis risk and is thought to increase reactivity to subsequent minor daily social stressors. Virtual reality allows controlled experimental exposure to virtual social stressors. To investigate the interplay between social adversity during early life, cell numbers of NK cells and T helper subsets and social stress reactivity in relation to psychosis liability. Circulating numbers of Th1, Th2, Th17, T regulator and NK cells were determined using flow cytometry in 80 participants with low psychosis liability (46 healthy controls and 34 siblings) and 53 participants with high psychosis liability (14 ultra-high risk (UHR) patients and 39 recent-onset psychosis patients), with and without the experience of childhood trauma. We examined if cell numbers predicted subjective stress when participants were exposed to social stressors (crowdedness, hostility and being part of an ethnic minority) in a virtual reality environment. There were no significant group differences in Th1, Th2, Th17, T regulator and NK cell numbers between groups with a high or low liability for psychosis. However, in the high psychosis liability group, childhood trauma was associated with increased Th17 cell numbers (p = 0.028). Moreover, in the high psychosis liability group increased T regulator and decreased NK cell numbers predicted stress experience during exposure to virtual social stressors (p = 0.015 and p = 0.009 for T regulator and NK cells, respectively). A deranged Th17/T regulator balance and a reduced NK cell number are associated intermediate biological factors in the relation childhood trauma, psychosis liability and social stress reactivity. Copyright © 2018 Elsevier Inc. All rights reserved.

Activated Tissue-Resident Mesenchymal Stromal Cells Regulate Natural Killer Cell Immune and Tissue-Regenerative Function.

PubMed

Petri, Robert Michael; Hackel, Alexander; Hahnel, Katrin; Dumitru, Claudia Alexandra; Bruderek, Kirsten; Flohe, Stefanie B; Paschen, Annette; Lang, Stephan; Brandau, Sven

2017-09-12

The interaction of mesenchymal stromal cells (MSCs) with natural killer (NK) cells is traditionally thought of as a static inhibitory model, whereby resting MSCs inhibit NK cell effector function. Here, we use a dynamic in vitro system of poly(I:C) stimulation to model the interaction of NK cells and tissue-resident MSCs in the context of infection or tissue injury. The experiments suggest a time-dependent system of regulation and feedback, where, at early time points, activated MSCs secrete type I interferon to enhance NK cell effector function, while at later time points TGF-β and IL-6 limit NK cell effector function and terminate inflammatory responses by induction of a regulatory senescent-like NK cell phenotype. Importantly, feedback of these regulatory NK cells to MSCs promotes survival, proliferation, and pro-angiogenic properties. Our data provide additional insight into the interaction of stromal cells and innate immune cells and suggest a model of time-dependent MSC polarization and licensing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity

PubMed Central

Kim, Won Sam; Kim, Mi Jeong; Kim, Dong Oh; Byun, Jae-Eun; Huy, Hangsak; Song, Hae Young; Park, Young-Jun; Kim, Tae-Don; Yoon, Suk Ran; Choi, Eun-Ji; Jung, Haiyoung; Choi, Inpyo

2017-01-01

Suppressor of cytokine signaling (SOCS) proteins are negative regulators of cytokine responses. Although recent reports have shown regulatory roles for SOCS proteins in innate and adaptive immunity, their roles in natural killer (NK) cell development are largely unknown. Here, we show that SOCS2 is involved in NK cell development. SOCS2−/− mice showed a high frequency of NK cells in the bone marrow and spleen. Knockdown of SOCS2 was associated with enhanced differentiation of NK cells in vitro, and the transplantation of hematopoietic stem cells (HSCs) into congenic mice resulted in enhanced differentiation in SOCS2−/− HSCs. We found that SOCS2 could inhibit Janus kinase 2 (JAK2) activity and JAK2-STAT5 signaling pathways via direct interaction with JAK2. Furthermore, SOCS2−/− mice showed a reduction in lung metastases and an increase in survival following melanoma challenge. Overall, our findings suggest that SOCS2 negatively regulates the development of NK cells by inhibiting JAK2 activity via direct interaction. PMID:28383049

Augmented IFN-γ and TNF-α Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL-10

PubMed Central

Bui, Vickie T.; Tseng, Han-Ching; Kozlowska, Anna; Maung, Phyu Ou; Kaur, Kawaljit; Topchyan, Paytsar; Jewett, Anahid

2015-01-01

Our previous reports demonstrated that the magnitude of natural killer (NK) cell-mediated cytotoxicity correlate directly with the stage and level of differentiation of tumor cells. In addition, we have shown previously that activated NK cells inhibit growth of cancer cells through induction of differentiation, resulting in the resistance of tumor cells to NK cell-mediated cytotoxicity through secreted cytokines, as well as direct NK-tumor cell contact. In this report, we show that in comparison to IL-2 + anti-CD16mAb-treated NK cells, activation of NK cells by probiotic bacteria (sAJ2) in combination with IL-2 and anti-CD16mAb substantially decreases tumor growth and induces maturation, differentiation, and resistance of oral squamous cancer stem cells, MIA PaCa-2 stem-like/poorly differentiated pancreatic tumors, and healthy stem cells of apical papillae through increased secretion of IFN-γ and TNF-α, as well as direct NK-tumor cell contact. Tumor resistance to NK cell-mediated killing induced by IL-2 + anti-CD16mAb + sAJ2-treated NK cells is induced by combination of IFN-γ and TNF-α since antibodies to both, and not each cytokine alone, were able to restore tumor sensitivity to NK cells. Increased surface expression of CD54, B7H1, and MHC-I on NK-differentiated tumors was mediated by IFN-γ since the addition of anti-IFN-γ abolished their increase and restored the ability of NK cells to trigger cytokine and chemokine release; whereas differentiated tumors inhibited cytokine release by the NK cells. Monocytes synergize with NK cells in the presence of probiotic bacteria to induce regulated differentiation of stem cells through secretion of IL-10 resulting in resistance to NK cell-mediated cytotoxicity and inhibition of cytokine release. Therefore, probiotic bacteria condition activated NK cells to provide augmented differentiation of cancer stem cells resulting in inhibition of tumor growth, and decreased inflammatory cytokine release. PMID:26697005

Synergistic inhibition of natural killer cells by the nonsignaling molecule CD94.

PubMed

Cheent, Kuldeep S; Jamil, Khaleel M; Cassidy, Sorcha; Liu, Mengya; Mbiribindi, Berenice; Mulder, Arend; Claas, Frans H J; Purbhoo, Marco A; Khakoo, Salim I

2013-10-15

Peptide selectivity is a feature of inhibitory receptors for MHC class I expressed by natural killer (NK) cells. CD94-NKG2A operates in tandem with the polymorphic killer cell Ig-like receptors (KIR) and Ly49 systems to inhibit NK cells. However, the benefits of having two distinct inhibitory receptor-ligand systems are not clear. We show that noninhibitory peptides presented by HLA-E can augment the inhibition of NKG2A(+) NK cells mediated by MHC class I signal peptides through the engagement of CD94 without a signaling partner. Thus, CD94 is a peptide-selective NK cell receptor, and NK cells can be regulated by nonsignaling interactions. We also show that KIR(+) and NKG2A(+) NK cells respond with differing stoichiometries to MHC class I down-regulation. MHC-I-bound peptide functions as a molecular rheostat controlling NK cell function. Selected peptides which in isolation do not inhibit NK cells can have different effects on KIR and NKG2A receptors. Thus, these two inhibitory systems may complement each other by having distinct responses to bound peptide and surface levels of MHC class I.

Mouse natural killer cell development and maturation are differentially regulated by SHIP-1.

PubMed

Banh, Cindy; Miah, S M Shahjahan; Kerr, William G; Brossay, Laurent

2012-11-29

The SH2-containing inositol phosphatase-1 (SHIP-1) is a 5' inositol phosphatase known to negatively regulate the product of phosphoinositide-3 kinase (PI3K), phosphatidylinositol-3.4,5-trisphosphate. SHIP-1 can be recruited to a large number of inhibitory receptors expressed on natural killer (NK) cells. However, its role in NK cell development, maturation, and functions is not well defined. In this study, we found that the absence of SHIP-1 results in a loss of peripheral NK cells. However, using chimeric mice we demonstrated that SHIP-1 expression is not required intrinsically for NK cell lineage development. In contrast, SHIP-1 is required cell autonomously for NK cell terminal differentiation. These findings reveal both a direct and indirect role for SHIP-1 at different NK cell development checkpoints. Notably, SHIP-1-deficient NK cells display an impaired ability to secrete IFN-γ during cytokine receptor-mediated responses, whereas immunoreceptor tyrosine-based activation motif containing receptor-mediated responses is not affected. Taken together, our results provide novel insights on how SHIP-1 participates in the development, maturation, and effector functions of NK cells.

Synergistic inhibition of natural killer cells by the nonsignaling molecule CD94

PubMed Central

Cheent, Kuldeep S.; Jamil, Khaleel M.; Cassidy, Sorcha; Liu, Mengya; Mbiribindi, Berenice; Mulder, Arend; Claas, Frans H. J.; Purbhoo, Marco A.; Khakoo, Salim I.

2013-01-01

Peptide selectivity is a feature of inhibitory receptors for MHC class I expressed by natural killer (NK) cells. CD94–NKG2A operates in tandem with the polymorphic killer cell Ig-like receptors (KIR) and Ly49 systems to inhibit NK cells. However, the benefits of having two distinct inhibitory receptor–ligand systems are not clear. We show that noninhibitory peptides presented by HLA-E can augment the inhibition of NKG2A+ NK cells mediated by MHC class I signal peptides through the engagement of CD94 without a signaling partner. Thus, CD94 is a peptide-selective NK cell receptor, and NK cells can be regulated by nonsignaling interactions. We also show that KIR+ and NKG2A+ NK cells respond with differing stoichiometries to MHC class I down-regulation. MHC-I–bound peptide functions as a molecular rheostat controlling NK cell function. Selected peptides which in isolation do not inhibit NK cells can have different effects on KIR and NKG2A receptors. Thus, these two inhibitory systems may complement each other by having distinct responses to bound peptide and surface levels of MHC class I. PMID:24082146

Impaired natural killer cell self-education and "missing-self" responses in Ly49-deficient mice.

PubMed

Bélanger, Simon; Tu, Megan M; Rahim, Mir Munir Ahmed; Mahmoud, Ahmad B; Patel, Rajen; Tai, Lee-Hwa; Troke, Angela D; Wilhelm, Brian T; Landry, Josette-Renée; Zhu, Qinzhang; Tung, Kenneth S; Raulet, David H; Makrigiannis, Andrew P

2012-07-19

Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.

Up-regulated expression of substance P in CD8+ T cells and NK1R on monocytes of atopic dermatitis.

PubMed

Zhang, Zenan; Zheng, Wenjiao; Xie, Hua; Chai, Ruonan; Wang, Junling; Zhang, Huiyun; He, Shaoheng

2017-05-01

Large numbers of CD8 + T cells were observed in atopic dermatitis (AD) skin, and monocytes from AD patients showed increased prostaglandin E2 production. However, little is known about the expression of substance P (SP) and its receptor NK1R in blood leukocytes of patients with AD. To explore the expression of SP and NK1R in leukocytes of AD and the influence of allergens on SP and NK1R expression. The expression levels of SP and NK1R in patients with AD were examined by flow cytometry, ELISA and a mouse AD model. The plasma SP level was 4.9-fold higher in patients with AD than in HC subjects. Both the percentage of SP expression in the population and mean fluorescence intensity (MFI) of SP expression were elevated in CD8 + T cells in the blood of AD patients. However, both the CD14 + NK1R + population and MFI of NK1R expression on CD14 + cells were enhanced in the blood of AD patients. Allergens ASWE, HDME and PPE failed to up-regulate SP expression in CD8 + T cells. However, allergens ASWE and HDME both enhanced NK1R expression on CD14 + blood leukocytes regardless of AD or HC subjects. OVA-sensitized AD mice showed an elevated proportion and MFI of SP-expressing CD8 + T cells in the blood, which agrees with the SP expression situation in human AD blood. Injection of SP into mouse skin did not up-regulate NK1R expression on monocytes. An elevated plasma SP level, up-regulated expression of SP and NK1R indicate that the SP/NK1R complex is important in the development of AD. Therefore, SP and NK1R antagonist or blocker agents may help to treat patients with AD. Trial registration Registration number: ChiCTR-BOC-16010279; Registration date: Dec., 28, 2016; retrospectively registered.

Resident Peritoneal NK cells

PubMed Central

Gonzaga, Rosemary; Matzinger, Polly; Perez-Diez, Ainhoa

2011-01-01

Here we describe a new population of NK cells that reside in the normal, un-inflamed peritoneal cavity. Phenotypically, they share some similarities with the small population of CD49b negative, CD27 positive immature splenic NK cells, and liver NK cells but differ in their expression of CD62L, TRAIL and EOMES. Functionally, the peritoneal NK cells resemble the immature splenic NK cells in their production of IFN-γ, GM-CSF and TNF-α and in the killing of YAC-1 target cells. We also found that the peritoneum induces different behavior in mature and immature splenic NK cells. When transferred intravenously into RAGγcKO mice, both populations undergo homeostatic proliferation in the spleen, but only the immature splenic NK cells, are able to reach the peritoneum. When transferred directly into the peritoneum, the mature NK cells survive but do not divide, while the immature NK cells proliferate profusely. These data suggest that the peritoneum is not only home to a new subset of tissue resident NK cells but that it differentially regulates the migration and homeostatic proliferation of immature versus mature NK cells. PMID:22079985

Therapeutic Potential and Challenges of Natural Killer Cells in Treatment of Solid Tumors

PubMed Central

Gras Navarro, Andrea; Björklund, Andreas T.; Chekenya, Martha

2015-01-01

Natural killer (NK) cells are innate lymphoid cells that hold tremendous potential for effective immunotherapy for a broad range of cancers. Due to the mode of NK cell killing, requiring one-to-one target engagement and site-directed release of cytolytic granules, the therapeutic potential of NK cells has been most extensively explored in hematological malignancies. However, their ability to precisely kill antibody coated cells, cancer stem cells, and genotoxically altered cells, while maintaining tolerance to healthy cells makes them appealing therapeutic effectors for all cancer forms, including metastases. Due to their release of pro-inflammatory cytokines, NK cells may potently reverse the anti-inflammatory tumor microenvironment (TME) and augment adaptive immune responses by promoting differentiation, activation, and/or recruitment of accessory immune cells to sites of malignancy. Nevertheless, integrated and coordinated mechanisms of subversion of NK cell activity against the tumor and its microenvironment exist. Although our understanding of the receptor ligand interactions that regulate NK cell functionality has evolved remarkably, the diversity of ligands and receptors is complex, as is their mechanistic foundations in regulating NK cell function. In this article, we review the literature and highlight how the TME manipulates the NK cell phenotypes, genotypes, and tropism to evade tumor recognition and elimination. We discuss counter strategies that may be adopted to augment the efficacy of NK cell anti-tumor surveillance, the clinical trials that have been undertaken so far in solid malignancies, critically weighing the challenges and opportunities with this approach. PMID:25972872

Uterine Natural Killer cells regulate endometrial bleeding in women and are suppressed by the progesterone receptor modulator asoprisnil

PubMed Central

Wilkens, Julia; Male, Victoria; Ghazal, Peter; Forster, Thorsten; Gibson, Douglas A.; Williams, Alistair RW; Brito-Mutunayagam, Savita L; Craigon, Marie; Lourenco, Paula; Cameron, Iain T; Chwalisz, Kristof; Moffett, Ashley; Critchley, Hilary OD

2013-01-01

Uterine NK cells (uNK) play a role in the regulation of placentation but their functions in non-pregnant endometrium are not understood. We have previously reported suppression of endometrial bleeding and alteration of spiral artery morphology in women exposed to asoprisnil, a progesterone receptor modulator. We now compare global endometrial gene expression in asoprisnil-treated versus control women, and we demonstrate a statistically significant reduction of genes in the IL-15 pathway, known to play a key role in uNK development and function. Suppression of IL-15 by asoprisnil was also observed at mRNA level (p<0.05), and immunostaining for NK cell marker CD56 revealed a striking reduction of uNK in asoprisnil-treated endometrium (p<0.001). IL-15 levels in normal endometrium are progesterone-responsive. Progesterone receptor (PR) positive stromal cells transcribe both IL-15 and IL-15RA. Thus, the response of stromal cells to progesterone will be to increase IL-15 trans-presentation to uNK, supporting their expansion and differentiation. In asoprisnil-treated endometrium, there is a marked down-regulation of stromal PR expression and virtual absence of uNK. These novel findings indicate that the IL-15 pathway provides a missing link in the complex interplay between endometrial stromal cells, uNK and spiral arteries affecting physiological and pathological endometrial bleeding. PMID:23913972

Viral antigen mediated NKp46 activation of NK cells results in tumor rejection via NK-DC crosstalk

PubMed Central

Chinnery, Fay; King, Catherine A.; Elliott, Tim; Bateman, Andrew R.; James, Edward

2012-01-01

Natural killer (NK) cells play a critical role in antitumor immunity, their activation being regulated through NK cell receptors. Although the endogenous ligands for these receptors are largely unknown, viral ligands have been identified. We investigated the ability of an activating NK receptor ligand derived from the mumps virus, haemagglutinin-neuraminidase (HN) to enhance NK activation against tumor cells. HN-expressing B16.OVA tumor cells induced stronger activation of NK cells compared with B16.OVA cells and also promoted dendritic cell (DC) activation toward a DC1 phenotype, in vitro. Moreover, incubation of DCs, NK cells and HN-expressing B16-OVA cells further enhanced NK cell activation through the NK-DC crosstalk, in a cell-to-cell contact- and IL-12-dependent fashion. Immunization of mice with HN-expressing B16-OVA cells resulted in > 85% survival rate after subsequent challenge with parental B16 or B16.OVA tumor cells. Tumor rejection was dependent on both NK and CD8+ T cells but not on CD4+ T cells, demonstrating induction of an effective adaptive immune response through innate immune cell activation. Our data indicate the potential of using robust NK cell activation, which through the NK-DC crosstalk stimulates effective antitumor responses, providing an alternate vaccine strategy. PMID:23162755

Distinct regulatory functions of SLP-76 and MIST in NK cell cytotoxicity and IFN-gamma production.

PubMed

Hidano, Shinya; Sasanuma, Hiroki; Ohshima, Keiko; Seino, Ken-ichiro; Kumar, Lalit; Hayashi, Katsuhiko; Hikida, Masaki; Kurosaki, Tomohiro; Taniguchi, Masaru; Geha, Raif S; Kitamura, Daisuke; Goitsuka, Ryo

2008-03-01

Activation of NK cells is triggered by multiple receptors. We demonstrate here that SLP-76 is required for CD16- and NKG2D-mediated NK cell cytotoxicity, while MIST negatively regulates these responses in an SLP-76-dependent manner. Exceptionally, MIST acts as a positive regulator of cytotoxicity against YAC-1 cells, although SLP-76 plays a more key role. SLP-76 acts as a dominant positive regulator for both NKG2D-mediated and YAC-1 cell-triggered IFN-gamma production. Although NKG2D-mediated IFN-gamma production depends on phospholipase C (PLC) gamma 2, YAC-1 cell-triggered IFN-gamma production is PLC gamma 2- and Syk/ZAP-70 independent and nuclear factor-kappa B mediated. SLP-76 is required for this process in the presence of MIST but is dispensable in the absence of MIST. Thus, YAC-1 cell-triggered NKG2D-independent IFN-gamma production appears to be regulated by SLP-76-dependent and -independent pathways, in which the latter is negatively regulated by MIST. Taken together, these results suggest that SLP-76 and MIST distinctly but interactively regulate NK cell cytotoxicity and IFN-gamma production.

Histaminergic regulation of natural killer cell-mediated clearance of tumour cells in mice.

PubMed

Asea, A; Hermodsson, S; Hellstrand, K

1996-01-01

Treatment of Swiss albino mice with histamine enhanced the clearance of natural killer (NK)-cell sensitive YAC-1 lymphoma and B16/F10 melanoma cells from lung tissue in vivo, but did not affect the elimination of NK-cell-insensitive P815 mastocytoma cells. The effect of histamine was apparently mediated by H2-type histamine receptors (H2R) since it was blocked by ranitidine, and H2R antagonist. Histamine did not affect clearance of tumour cells in animals depleted of NK cells in vivo by treatment with antibodies to asialo-GM1 or NK1.1. The effect of histamine was time-dependent: pretreatment with histamine for 3 h significantly augmented the clearance of YAC-1 cells, whereas, pretreatment with histamine for 5 min was ineffective. Histamine potentiated the anti-tumour properties of NK-cell activators such as interleukin-2 (IL-2) or interferon-alpha (IFN-alpha) in vivo. None of these lymphokines significantly affected the clearance of YAC-1 cells unless animals were concomitantly treated with histamine. Treatment with ranitidine alone reduced the in vivo clearance of YAC-1 cells from lungs but did not affect the clearance of NK-cell-insensitive P815 cells. Effects of ranitidine on NK-cell function in vivo were not shared by a chemical control to ranitidine, AH20239AA, thus indicating that the inhibition of NK-cells results from H2R antagonism rather than non-specific toxicity. It is concluded that histaminergic mechanisms may be involved in the regulation of NK cell function in vivo.

NK cell-derived IL-10 is critical for DC-NK cell dialogue at the maternal-fetal interface.

PubMed

Blois, Sandra M; Freitag, Nancy; Tirado-González, Irene; Cheng, Shi-Bin; Heimesaat, Markus M; Bereswill, Stefan; Rose, Matthias; Conrad, Melanie L; Barrientos, Gabriela; Sharma, Surendra

2017-05-19

DC-NK cell interactions are thought to influence the development of maternal tolerance and de novo angiogenesis during early gestation. However, it is unclear which mechanism ensures the cooperative dialogue between DC and NK cells at the feto-maternal interface. In this article, we show that uterine NK cells are the key source of IL-10 that is required to regulate DC phenotype and pregnancy success. Upon in vivo expansion of DC during early gestation, NK cells expressed increased levels of IL-10. Exogenous administration of IL-10 was sufficient to overcome early pregnancy failure in dams treated to achieve simultaneous DC expansion and NK cell depletion. Remarkably, DC expansion in IL-10 -/- dams provoked pregnancy loss, which could be abrogated by the adoptive transfer of IL-10 +/+ NK cells and not by IL-10 -/- NK cells. Furthermore, the IL-10 expressing NK cells markedly enhanced angiogenic responses and placental development in DC expanded IL-10 -/- dams. Thus, the capacity of NK cells to secrete IL-10 plays a unique role facilitating the DC-NK cell dialogue during the establishment of a healthy gestation.

Location and cellular stages of NK cell development

PubMed Central

Yu, Jianhua; Freud, Aharon G.; Caligiuri, Michael A

2013-01-01

The identification of distinct tissue-specific natural killer (NK) cell populations that apparently mature from local precursor populations has brought new insight into the diversity and developmental regulation of this important lymphoid subset. NK cells provide a necessary link between the early (innate) and late (adaptive) immune responses to infection. Gaining a better understanding of the processes that govern NK cell development should allow us to better harness NK cell functions in multiple clinical settings as well as to gain further insight into how these cells undergo malignant transformation. In this review, we summarize recent advances in understanding sites and cellular stages of NK cell development in humans and mice. PMID:24055329

Regulation of Mouse NK Cell Development and Function by Cytokines

PubMed Central

Marçais, Antoine; Viel, Sébastien; Grau, Morgan; Henry, Thomas; Marvel, Jacqueline; Walzer, Thierry

2013-01-01

Natural Killer (NK) cells are innate lymphocytes with an important role in the early defense against intracellular pathogens and against tumors. Like other immune cells, almost every aspects of their biology are regulated by cytokines. Interleukin (IL)-15 is pivotal for their development, homeostasis, and activation. Moreover, numerous other activating or inhibitory cytokines such as IL-2, IL-4, IL-7, IL-10, IL-12, IL-18, IL-21, Transforming growth factor-β (TGFβ) and type I interferons regulate their activation and their effector functions at different stages of the immune response. In this review we summarize the current understanding on the effect of these different cytokines on NK cell development, homeostasis, and functions during steady-state or upon infection by different pathogens. We try to delineate the cellular sources of these cytokines, the intracellular pathways they trigger and the transcription factors they regulate. We describe the known synergies or antagonisms between different cytokines and highlight outstanding questions in this field of investigation. Finally, we discuss how a better knowledge of cytokine action on NK cells could help improve strategies to manipulate NK cells in different clinical situations. PMID:24376448

Haemagglutinin-neuraminidase from HPIV3 mediates human NK regulation of T cell proliferation via NKp44 and NKp46.

PubMed

McQuaid, Samantha; Loughran, Sinead; Power, Patrick; Maguire, Paula; Walls, Dermot; Grazia Cusi, Maria; Orvell, Claes; Johnson, Patricia

2018-06-01

HPIV3 is a respiratory virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. Currently there is no effective vaccine or anti-viral therapy for this virus. Studies have suggested that poor T cell proliferation following HPIV3 infection is responsible for impaired immunological memory associated with this virus. We have previously demonstrated that NK cells mediate regulation of T cell proliferation during HPIV3 infection. Here we add to these studies by demonstrating that the regulation of T cell proliferation during HPIV3 infection is mediated via NK receptors NKp44 and NKp46 and involves the surface glycoprotein haemagglutinin-neuraminidase but not the fusion protein of the virus. These studies extend our knowledge of the regulatory repertoire of NK cells and provide mechanistic insights which may explain reoccurring failures of vaccines against this virus.

Antiviral activity of NK 1.1+ natural killer cells in C57BL/6 scid mice infected with murine cytomegalovirus.

PubMed

Welsh, R M; O'Donnell, C L; Shultz, L D

1994-01-01

The activation, proliferation, and antiviral effects of natural killer (NK) cells were examined in a newly developed stock of mice, C57BL/6JSz mice homozygous for the severe combined immunodeficiency (scid) mutation. These mice lack functional T and B cells and express the NK 1.1 alloantigen. Such NK 1.1 expression facilitates the analysis of NK cells and their depletion in vivo with a monoclonal anti-NK 1.1 antibody. These mice, therefore, provide an excellent model to examine unambiguously the interactions between viral infections and NK cells in a system devoid of adaptive immune response mechanisms. Here we show that murine cytomegalovirus (MCMV) and lymphocytic choriomeningitis virus (LCMV) infections resulted in profound levels of NK cell activation. NK cells also proliferated greatly in response to LCMV but generally to a lesser degree in response to MCMV. Depletion of the NK cell activity in vivo caused substantial increases in MCMV synthesis and MCMV-induced pathology. These results further support the concept that NK cells are major regulators of MCMV pathogenesis.

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

Regulated Norepinephrine Transporter Interaction with the Neurokinin-1 Receptor Establishes Transporter Subcellular Localization*

PubMed Central

Arapulisamy, Obulakshmi; Mannangatti, Padmanabhan; Jayanthi, Lankupalle D.

2013-01-01

Neurokinin-1 receptor (NK1R) mediates down-regulation of human norepinephrine (NE) transporter (hNET) via protein kinase C (PKC). However, native NET regulation by NK1R and the mechanism by which NK1R targets NET among other potential effectors are unknown. Effect of NK1R activation on native NET regulation and NET/NK1R interaction were studied using rat brain synaptosomes expressing native NET and NK1R as well as human placental trophoblast (HTR) cells coexpressing WT-hNET or NK1R/PKC-resistant hNET-T258A,S259A double mutant (NET-DM) and hNK1R. The selective NK1R agonist, GR73632, and Substance-P (SP) inhibited NE transport and reduced plasma membrane expression of NET and NK1R. Pretreatment with the NK1R antagonist, EMEND (aprepitant) prevented these NK1R-mediated effects. Immunoprecipitation experiments showed that NET forms stable complexes with NK1R. In HTR cells, combined biotinylation and immunoprecipitation studies revealed plasma membrane localization of NET·NK1R complexes. Receptor activation resulted in the internalization of NET·NK1R complexes. Lipid raft and immunoprecipitation analyses revealed the presence of NET·NK1R complexes exclusively in non-raft membrane fractions under basal/unstimulated conditions. However, NK1R activation led to translocation of NET·NK1R complexes to raft-rich membrane fractions. Importantly, PKCα was found in association with raft-localized NET following SP treatment. Similar to WT-NET, PKC-resistant NET-DM was found in association with NK1R exclusively in non-raft fractions. However, SP treatment failed to translocate NET-DM·NK1R complexes from non-raft fractions to raft fractions. Collectively, these results suggest that NK1R forms physical complexes with NET and that the receptor-mediated Thr258 + Ser259 motif-dependent translocation of NET·NK1R complexes into raft-rich microdomains facilitates NET/NK1R interaction with PKCα to coordinate spatially restricted NET regulation. PMID:23979140

Regulated norepinephrine transporter interaction with the neurokinin-1 receptor establishes transporter subcellular localization.

PubMed

Arapulisamy, Obulakshmi; Mannangatti, Padmanabhan; Jayanthi, Lankupalle D

2013-10-04

Neurokinin-1 receptor (NK1R) mediates down-regulation of human norepinephrine (NE) transporter (hNET) via protein kinase C (PKC). However, native NET regulation by NK1R and the mechanism by which NK1R targets NET among other potential effectors are unknown. Effect of NK1R activation on native NET regulation and NET/NK1R interaction were studied using rat brain synaptosomes expressing native NET and NK1R as well as human placental trophoblast (HTR) cells coexpressing WT-hNET or NK1R/PKC-resistant hNET-T258A,S259A double mutant (NET-DM) and hNK1R. The selective NK1R agonist, GR73632, and Substance-P (SP) inhibited NE transport and reduced plasma membrane expression of NET and NK1R. Pretreatment with the NK1R antagonist, EMEND (aprepitant) prevented these NK1R-mediated effects. Immunoprecipitation experiments showed that NET forms stable complexes with NK1R. In HTR cells, combined biotinylation and immunoprecipitation studies revealed plasma membrane localization of NET·NK1R complexes. Receptor activation resulted in the internalization of NET·NK1R complexes. Lipid raft and immunoprecipitation analyses revealed the presence of NET·NK1R complexes exclusively in non-raft membrane fractions under basal/unstimulated conditions. However, NK1R activation led to translocation of NET·NK1R complexes to raft-rich membrane fractions. Importantly, PKCα was found in association with raft-localized NET following SP treatment. Similar to WT-NET, PKC-resistant NET-DM was found in association with NK1R exclusively in non-raft fractions. However, SP treatment failed to translocate NET-DM·NK1R complexes from non-raft fractions to raft fractions. Collectively, these results suggest that NK1R forms physical complexes with NET and that the receptor-mediated Thr(258) + Ser(259) motif-dependent translocation of NET·NK1R complexes into raft-rich microdomains facilitates NET/NK1R interaction with PKCα to coordinate spatially restricted NET regulation.

NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing

PubMed Central

Hsu, Hsiang-Ting; Viswanath, Dixita I.; Önfelt, Björn

2016-01-01

Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein- and integrin signal–dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector–target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific “bystander” killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells. PMID:27903610

Sorafenib paradoxically activates the RAS/RAF/ERK pathway in polyclonal human NK cells during expansion and thereby enhances effector functions in a dose- and time-dependent manner.

PubMed

Lohmeyer, J; Nerreter, T; Dotterweich, J; Einsele, H; Seggewiss-Bernhardt, R

2018-03-24

Natural killer (NK) cells play a major role in host immunity against leukaemia and lymphoma. However, clinical trials applying NK cells have not been as efficient as hoped for. Patients treated with rapidly accelerated fibrosarcoma (RAF) inhibitors exhibit increased tumour infiltration by immune cells, suggesting that a combination of RAF inhibitors with immunotherapy might be beneficial. As mitogen-activated protein kinases (MAPKs) such as raf-1 proto-oncogene, serine/threonine kinase (CRAF) regulate NK cell functions, we performed an in-vitro investigation on the potential of clinically relevant short-acting tyrosine kinase inhibitors (TKIs) as potential adjuvants for NK cell therapy: NK cells from healthy human blood donors were thus treated with sorafenib, sunitinib or the pan-RAF inhibitor ZM336372 during ex-vivo expansion. Functional outcomes assessed after washout of the drugs included cytokine production, degranulation, cytotoxicity, apoptosis induction and signal transduction with/without target cell contact. Paradoxically, sorafenib enhanced NK cell effector functions in a time- and dose-dependent manner by raising the steady-state activation level. Of note, this did not lead to NK cell exhaustion, but enhanced activity against target cells such as K562 or Daudis mediated via the RAS/RAF/extracellular-regulated kinase (ERK) pathway, but not via protein kinase B (AKT). Our data will pave the path to develop a rationale for the considered use of RAF inhibitors such as sorafenib for pre-activation in NK cell-based adoptive immune therapy. © 2018 British Society for Immunology.

Up-regulation of NKG2A inhibitory receptor on circulating NK cells contributes to transfusion-induced immunodepression in patients with β-thalassemia major.

PubMed

Zou, Yong; Song, Zhi-Xing; Lu, Ying; Liang, Xiao-Li; Yuan, Qing; Liao, Si-Hong; Bao, Jun-Jie

2016-08-01

Accumulating evidence has shown that allogeneic blood transfusions can induce significant immunosuppression in recipients, and thereby increase the risk of postoperative infection and/or tumor relapse. Although it is well known that natural killer (NK) cells are responsible for the immunodepression effects of transfusion, the underlying mechanisms remain obscure. In this study, we investigated the role of NK cells in transfusion-induced immunodepression in β-thalassemia major. The proportion of circulating NK cells and the expression of NK receptors (NKG2A, CD158a, NKP30, NKP46 and NKG2D) as well as CD107a were detected by multicolor flow cytometry. IFN-γ production by circulating NK cells was detected by intracellular cytokine staining. Our results showed that the proportion and cytotoxicity (CD107a expression) of circulating NK cells in transfusion-dependent β-thalassemia major patients were remarkably lower than those of β-thalassemia minor patients or healthy volunteers. Expression of NKG2A inhibitory receptor on circulating NK cells in patients with β-thalassemia major was remarkably up-regulated, but there were no significant differences in the expression levels of NKP30, NKP46, NKG2D, CD158a and IFN-γ. These results indicate NKG2A inhibitory receptor may play a key role in transfusion-induced immunodepression of NK cells in patients with β-thalassemia major.

Endometrial natural killer (NK) cells reveal a tissue-specific receptor repertoire.

PubMed

Feyaerts, D; Kuret, T; van Cranenbroek, B; van der Zeeuw-Hingrez, S; van der Heijden, O W H; van der Meer, A; Joosten, I; van der Molen, R G

2018-02-13

Is the natural killer (NK) cell receptor repertoire of endometrial NK (eNK) cells tissue-specific? The NK cell receptor (NKR) expression profile in pre-pregnancy endometrium appears to have a unique tissue-specific phenotype, different from that found in NK cells in peripheral blood, suggesting that these cells are finely tuned towards the reception of an allogeneic fetus. NK cells are important for successful pregnancy. After implantation, NK cells encounter extravillous trophoblast cells and regulate trophoblast invasion. NK cell activity is amongst others regulated by C-type lectin heterodimer (CD94/NKG2) and killer cell immunoglobulin-like (KIR) receptors. KIR expression on decidual NK cells is affected by the presence of maternal HLA-C and biased towards KIR2D expression. However, little is known about NKR expression on eNK cells prior to pregnancy. In this study, matched peripheral and menstrual blood (a source of endometrial cells) was obtained from 25 healthy females with regular menstrual cycles. Menstrual blood was collected during the first 36 h of menstruation using a menstrual cup, a non-invasive technique to obtain endometrial cells. KIR and NKG2 receptor expression on eNK cells was characterized by 10-color flow cytometry, and compared to matched pbNK cells of the same female. KIR and HLA-C genotypes were determined by PCR-SSOP techniques. Anti-CMV IgG antibodies in plasma were measured by chemiluminescence immunoassay. KIR expression patterns of eNK cells collected from the same female do not differ over consecutive menstrual cycles. The percentage of NK cells expressing KIR2DL2/L3/S2, KIR2DL3, KIR2DL1, LILRB1 and/or NKG2A was significantly higher in eNK cells compared to pbNK cells, while no significant difference was observed for NKG2C, KIR2DL1/S1, and KIR3DL1. The NKR repertoire of eNK cells was clearly different from pbNK cells, with eNK cells co-expressing more than three NKR simultaneously. In addition, outlier analysis revealed 8 and 15 NKR subpopulation expansions in eNK and pbNK cells, respectively. In contrast to the pbNK cell population, the expansions present in the eNK cell population were independent of CMV status and HLA-C genotype. Moreover, the typical NKG2C imprint induced by CMV infection on pbNK cells was not observed on eNK cells from the same female, suggesting a rapid local turnover of eNK cells and/or a distinct licensing process. Based on our previous work and the parameters studied here, menstrual blood-derived eNK cells closely resemble biopsy-derived eNK cells. However, sampling is not done at the exact same time during the menstrual cycle, and therefore we cannot exclude some, as yet undetected, differences. Our data reveals that NK cells in the pre-implantation endometrium appear to have a dedicated tissue-specific phenotype, different from NK cells in peripheral blood. This may indicate that eNK cells are finely tuned to receive an allogeneic fetus. Studying the endometrial NKR repertoire of women with pregnancy related problems could provide clues to understand the pathogenesis of pregnancy complications. No external funding was obtained for the present study. None of the authors has any conflict of interest to declare. NA. © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

PTEN Is a Negative Regulator of NK Cell Cytolytic Function

PubMed Central

Briercheck, Edward L.; Trotta, Rossana; Chen, Li; Hartlage, Alex S.; Cole, Jordan P.; Cole, Tyler D.; Mao, Charlene; Banerjee, Pinaki P.; Hsu, Hsiang-Ting; Mace, Emily M.; Ciarlariello, David; Mundy-Bosse, Bethany L.; Garcia-Cao, Isabel; Scoville, Steven D.; Yu, Lianbo; Pilarski, Robert; Carson, William E.; Leone, Gustavo; Pandolfi, Pier Paolo; Yu, Jianhua; Orange, Jordan S.; Caligiuri, Michael A.

2015-01-01

Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood, the more mature CD56dim NK cell efficiently kills malignant targets at rest, whereas the less mature CD56bright NK cells cannot. In this study, we show that resting CD56bright NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56dim NK cells. Consistent with this, forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity, and loss of PTEN in CD56bright NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell–activating and inhibitory receptor expression yet, as in humans, did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell’s ability to organize immunological synapse components including decreases in actin accumulation, polarization of the microtubule organizing center, and the convergence of cytolytic granules. In summary, our data suggest that PTEN normally works to limit the NK cell’s PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56bright NK cell to the cytolytic CD56dim NK cells. PMID:25595786

Salivary Gland NK Cells Are Phenotypically and Functionally Unique

PubMed Central

Brossay, Laurent

2011-01-01

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 Treg 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. PMID:21249177

Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity.

PubMed

Schettini, Jorge; Kidiyoor, Amritha; Besmer, Dahlia M; Tinder, Teresa L; Roy, Lopamudra Das; Lustgarten, Joseph; Gendler, Sandra J; Mukherjee, Pinku

2012-11-01

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.

Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity

PubMed Central

Schettini, Jorge; Kidiyoor, Amritha; Besmer, Dahlia M.; Tinder, Teresa L.; Roy, Lopamudra Das; Lustgarten, Joseph; Gendler, Sandra J.

2013-01-01

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo. PMID:22543528

The hepatocyte growth factor antagonist NK4 inhibits indoleamine-2,3-dioxygenase expression via the c-Met-phosphatidylinositol 3-kinase-AKT signaling pathway

PubMed Central

WANG, DONGDONG; SAGA, YASUSHI; SATO, NAOTO; NAKAMURA, TOSHIKAZU; TAKIKAWA, OSAMU; MIZUKAMI, HIROAKI; MATSUBARA, SHIGEKI; FUJIWARA, HIROYUKI

2016-01-01

Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme involved in tumor malignancy. However, the regulatory mechanism underlying its involvement remains largely uncharacterized. The present study aimed to investigate the hypothesis that NK4, an antagonist of hepatocyte growth factor (HGF), can regulate IDO and to characterize the signaling mechanism involved. Following successful transfection of the human ovarian cancer cell line SKOV-3 (which constitutively expresses IDO) with an NK4 expression vector, we observed that NK4 expression suppressed IDO expression; furthermore, NK4 expression did not suppress cancer cell growth in vitro [in the absence of natural killer (NK) cells], but did influence tumor growth in vivo. In addition, NK4 enhanced the sensitivity of cancer cells to NK cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. In an effort to clarify the mechanisms by which NK4 interacts with IDO, we performed investigations utilizing various biochemical inhibitors. The results of these investigations were as follows. First, c-Met (a receptor of HGF) tyrosine kinase inhibitor PHA-665752, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 both suppress IDO expression. Second, enhanced expression of PTEN (a known tumor suppressor) via negative regulation within a PI3K-AKT pathway, inhibits IDO expression. Conversely, neither the MEK1/2 inhibitor U0126 nor the STAT3 inhibitor WP1066 affects IDO expression. These results suggest that NK4 inhibits IDO expression via a c-Met-PI3K-AKT signaling pathway. PMID:27082119

Natural Killer (NK)/melanoma cell interaction induces NK-mediated release of chemotactic High Mobility Group Box-1 (HMGB1) capable of amplifying NK cell recruitment

PubMed Central

Parodi, Monica; Pedrazzi, Marco; Cantoni, Claudia; Averna, Monica; Patrone, Mauro; Cavaletto, Maria; Spertino, Stefano; Pende, Daniela; Balsamo, Mirna; Pietra, Gabriella; Sivori, Simona; Carlomagno, Simona; Mingari, Maria Cristina; Moretta, Lorenzo; Sparatore, Bianca; Vitale, Massimo

2015-01-01

In this study we characterize a new mechanism by which Natural Killer (NK) cells may amplify their recruitment to tumors. We show that NK cells, upon interaction with melanoma cells, can release a chemotactic form of High Mobility Group Box-1 (HMGB1) protein capable of attracting additional activated NK cells. We first demonstrate that the engagement of different activating NK cell receptors, including those mainly involved in tumor cell recognition can induce the active release of HMGB1. Then we show that during NK-mediated tumor cell killing two HMGB1 forms are released, each displaying a specific electrophoretic mobility possibly corresponding to a different redox status. By the comparison of normal and perforin-defective NK cells (which are unable to kill target cells) we demonstrate that, in NK/melanoma cell co-cultures, NK cells specifically release an HMGB1 form that acts as chemoattractant, while dying tumor cells passively release a non-chemotactic HMGB1. Finally, we show that Receptor for Advanced Glycation End products is expressed by NK cells and mediates HMGB1-induced NK cell chemotaxis. Proteomic analysis of NK cells exposed to recombinant HMGB1 revealed that this molecule, besides inducing immediate chemotaxis, also promotes changes in the expression of proteins involved in the regulation of the cytoskeletal network. Importantly, these modifications could be associated with an increased motility of NK cells. Thus, our findings allow the definition of a previously unidentified mechanism used by NK cells to amplify their response to tumors, and provide additional clues for the emerging role of HMGB1 in immunomodulation and tumor immunity. PMID:26587323

SHIP1 intrinsically regulates NK cell signaling and education, resulting in tolerance of an MHC class I-mismatched bone marrow graft in mice.

PubMed

Gumbleton, Matthew; Vivier, Eric; Kerr, William G

2015-03-15

NK cells are an important component of host immune defense against malignancy and infection. NK cells are educated by MHC class I ligands to ensure self-tolerance while also promoting lytic competency against altered self and damaged self targets. However, the intracellular molecular events that culminate in tolerance and functional competency of educated NK cells remain undefined. Mice with germline deficiency in SHIP1 were shown to have a defective NK cell compartment. However, SHIP1 is expressed in all hematopoietic lineages, and consequently several hematolymphoid phenotypes have already been identified in certain cell types that are the result of SHIP1 deficiency in cells in separate and distinct lineages, that is, cell-extrinsic phenotypes. Thus, it was previously impossible to determine the NK cell-intrinsic role of SHIP1. In the present study, through the creation of an NK cell-specific deletion mouse model of SHIP1, we show that SHIP1 plays a profound NK lineage-intrinsic role in NK cell homeostasis, development, education, and cytokine production. Moreover, we show SHIP1 expression by NK cells is required for in vivo-mismatched bone marrow allograft rejection as well as for NK memory responses to hapten. Copyright © 2015 by The American Association of Immunologists, Inc.

Substance P inhibits natural killer cell cytotoxicity through the neurokinin-1 receptor.

PubMed

Monaco-Shawver, Linda; Schwartz, Lynnae; Tuluc, Florin; Guo, Chang-Jiang; Lai, Jian Ping; Gunnam, Satya M; Kilpatrick, Laurie E; Banerjee, Pinaki P; Douglas, Steven D; Orange, Jordan S

2011-01-01

SP is a potent neuroimmunomodulator that functions through ligating members of the neurokinin receptor family, one of which, NK1R, is widely expressed in immune cells. As in humans, circulating SP levels are increased in pathologic states associated with impairment of NK cell functions, such as depression and HIV infection, we hypothesized that SP has a direct, inhibitory effect upon NK cells. We have studied a clonal human NK cell line (YTS) as well as ex vivo human NK cells and have determined that truncated and full-length NK1R isoforms are expressed in and SP bound by ex vivo NK cells and the YTS NK cell line. Incubation of YTS cells with 10⁻⁶ M SP and ex vivo NK cells with 10⁻⁵ M SP inhibited cytotoxic ability by ∼20% and reduced degranulation. This inhibitory effect upon cytotoxicity was partially prevented by the NK1R antagonist CP96,345. The treatment of YTS or ex vivo NK cells with SP neither down-modulated NCR expression nor affected triggering receptor-induced NF-κB activation. Preincubation of YTS cells with SP, however, did abbreviate the typically prolonged intracellular calcium increase induced by target cell engagement and reduced triggering receptor-induced pERK. Thus, SP has the potential to regulate NK cell functions and acts downstream from neurokinin receptors to modulate NK cell activation signaling. This mechanism may contribute to impairment of NK cell function in certain disease states associated with increased circulating SP. Antagonism of this system may present an opportunity to augment NK cell function therapeutically in selected human diseases.

Next-generation sequencing identifies the natural killer cell microRNA transcriptome

PubMed Central

Fehniger, Todd A.; Wylie, Todd; Germino, Elizabeth; Leong, Jeffrey W.; Magrini, Vincent J.; Koul, Sunita; Keppel, Catherine R.; Schneider, Stephanie E.; Koboldt, Daniel C.; Sullivan, Ryan P.; Heinz, Michael E.; Crosby, Seth D.; Nagarajan, Rakesh; Ramsingh, Giridharan; Link, Daniel C.; Ley, Timothy J.; Mardis, Elaine R.

2010-01-01

Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g., granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates for mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes have been established, little is known about miRNAs in NK cells. Here, we used two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by quantitative real-time PCR (qRT-PCR) and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 21 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range and exhibit isomiR complexity, and a subset is differentially expressed following cytokine activation. Using these miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine activation. Furthermore, we demonstrate that miR-223 specifically targets the 3′ untranslated region of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology. PMID:20935160

PD-1 mediates functional exhaustion of activated NK cells in patients with Kaposi sarcoma

PubMed Central

Beldi-Ferchiou, Asma; Lambert, Marion; Dogniaux, Stéphanie; Vély, Frédéric; Vivier, Eric; Olive, Daniel; Dupuy, Stéphanie; Levasseur, Frank; Zucman, David; Lebbé, Céleste; Sène, Damien; Hivroz, Claire; Caillat-Zucman, Sophie

2016-01-01

Programmed Death-1 (PD-1), an inhibitory receptor expressed by activated lymphocytes, is involved in regulating T- and B-cell responses. PD-1 and its ligands are exploited by a variety of cancers to facilitate tumor escape through PD-1-mediated functional exhaustion of effector T cells. Here, we report that PD-1 is upregulated on Natural Killer (NK) cells from patients with Kaposi sarcoma (KS). PD-1 was expressed in a sub-population of activated, mature CD56dimCD16pos NK cells with otherwise normal expression of NK surface receptors. PD-1pos NK cells from KS patients were hyporesponsive ex vivo following direct triggering of NKp30, NKp46 or CD16 activating receptors, or short stimulation with NK cell targets. PD-1pos NK cells failed to degranulate and release IFNγ, but exogenous IL-2 or IL-15 restored this defect. That PD-1 contributed to NK cell functional impairment and was not simply a marker of dysfunctional NK cells was confirmed in PD-1-transduced NKL cells. In vitro, PD-1 was induced at the surface of healthy control NK cells upon prolonged contact with cells expressing activating ligands, i.e. a condition mimicking persistent stimulation by tumor cells. Thus, PD-1 appears to plays a critical role in mediating NK cell exhaustion. The existence of this negative checkpoint fine-tuning NK activation highlights the possibility that manipulation of the PD-1 pathway may be a strategy for circumventing tumor escape not only from the T cell-, but also the NK-cell mediated immune surveillance. PMID:27662664

Genetically Engineered Natural Killer Cells as a Means for Adoptive Tumor Immunotherapy.

PubMed

Michen, Susanne; Temme, Achim

2016-01-01

Natural killer (NK) cells are lymphoid cells of the innate immune system; they stand at the first defense line against viruses and transformed cells. NK cells use an array of germline-encoded activating and inhibitory receptors that sense virus-infected cells or malignant cells displaying altered surface expression of activating and inhibitory NK cell ligands. They exert potent cytotoxic responses to cellular targets and thus are candidate effector cells for immunotherapy of cancer. In particular, the genetic engineering of NK cells with chimeric antigen receptors (CARs) against surface-expressed tumor-associated antigens (TAAs) seems promising. In the allogeneic context, gene-modified NK cells compared to T cells may be superior because they are short-lived effector cells and do not cause graft-versus-host disease. Furthermore, their anti-tumoral activity can be augmented by combinatorial use with therapeutic antibodies, chemotherapeutics, and radiation. Today, efforts are being undertaken for large-scale NK-cell expansion and their genetic engineering for adoptive cell transfer. With the recent advances in understanding the complex biological interactions that regulate NK cells, it is expected that the genetic engineering of NK cells and a combinatorial blockade of immune evasion mechanisms are required to exploit the full potential of NK-cell-based immunotherapies.

MicroRNA-29b mediates altered innate immune development in acute leukemia

PubMed Central

Mundy-Bosse, Bethany L.; Scoville, Steven D.; Chen, Li; McConnell, Kathleen; Mao, Hsiaoyin C.; Ahmed, Elshafa H.; Zorko, Nicholas; Harvey, Sophia; Cole, Jordan; Zhang, Xiaoli; Costinean, Stefan; Croce, Carlo M.; Larkin, Karilyn; Byrd, John C.; Vasu, Sumithira; Blum, William; Yu, Jianhua; Freud, Aharon G.; Caligiuri, Michael A.

2016-01-01

Natural killer (NK) cells can have potent antileukemic activity following haplo-mismatched, T cell–depleted stem cell transplantations for the treatment of acute myeloid leukemia (AML), but they are not successful in eradicating de novo AML. Here, we have used a mouse model of de novo AML to elucidate the mechanisms by which AML evades NK cell surveillance. NK cells in leukemic mice displayed a marked reduction in the cytolytic granules perforin and granzyme B. Further, as AML progressed, we noted the selective loss of an immature subset of NK cells in leukemic mice and in AML patients. This absence was not due to elimination by cell death or selective reduction in proliferation, but rather to the result of a block in NK cell differentiation. Indeed, NK cells from leukemic mice and humans with AML showed lower levels of TBET and EOMES, transcription factors that are critical for terminal NK cell differentiation. Further, the microRNA miR-29b, a regulator of T-bet and EOMES, was elevated in leukemic NK cells. Finally, deletion of miR-29b in NK cells reversed the depletion of this NK cell subset in leukemic mice. These results indicate that leukemic evasion of NK cell surveillance occurs through miR-mediated dysregulation of lymphocyte development, representing an additional mechanism of immune escape in cancer. PMID:27775550

Canonical TGF-β Signaling Pathway Represses Human NK Cell Metabolism.

PubMed

Zaiatz-Bittencourt, Vanessa; Finlay, David K; Gardiner, Clair M

2018-06-15

Cytokines stimulate rapid metabolic changes in human NK cells, including increases in both glycolysis and oxidative phosphorylation pathways. However, how these are subsequently regulated is not known. In this study, we demonstrate that TGF-β can inhibit many of these metabolic changes, including oxidative phosphorylation, glycolytic capacity, and respiratory capacity. TGF-β also inhibited cytokine-induced expression of the transferrin nutrient receptor CD71. In contrast to a recent report on murine NK cells, TGF-β-mediated suppression of these metabolic responses did not involve the inhibition of the metabolic regulator mTORC1. Inhibition of the canonical TGF-β signaling pathway was able to restore almost all metabolic and functional responses that were inhibited by TGF-β. These data suggest that pharmacological inhibition of TGF-β could provide a metabolic advantage to NK cells that is likely to result in improved functional responses. This has important implications for NK cell-based cancer immunotherapies. Copyright © 2018 by The American Association of Immunologists, Inc.

NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors

PubMed Central

Anton, Olga M.; Vielkind, Susina; Peterson, Mary E.; Tagaya, Yutaka; Long, Eric O.

2015-01-01

IL-15 bound to the IL-15 receptor α chain (IL-15Rα) is presented in trans to cells bearing the IL-2 receptor β and γc chains. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor–ligand interactions. In this study, human NK cells were tested for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Rα. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIR–HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to down-regulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A+ cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Co-engagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15 dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis. PMID:26453750

Early Hematopoietic Zinc Finger Protein Prevents Tumor Cell Recognition by Natural Killer Cells1

PubMed Central

La Rocca, Rosanna; Fulciniti, Mariateresa; Lakshmikanth, Tadepally; Mesuraca, Maria; Ali, Talib Hassan; Mazzei, Valerio; Amodio, Nicola; Catalano, Lucio; Rotoli, Bruno; Ouerfelli, Ouathek; Grieco, Michele; Gulletta, Elio; Bond, Heather M.; Morrone, Giovanni; Ferrone, Soldano; Carbone, Ennio

2009-01-01

Early hematopoietic zinc finger/zinc finger protein 521 (EHZF/ZNF521) is a novel zinc finger protein expressed in hematopoietic stem and progenitor cells and is down-regulated during their differentiation. Its transcript is also abundant in some hematopoietic malignancies. Analysis of the changes in the antigenic profile of cells transfected with EHZF cDNA revealed up-regulation of HLA class I cell surface expression. This phenotypic change was associated with an increased level of HLA class I H chain, in absence of detectable changes in the expression of other Ag-processing machinery components. Enhanced resistance of target cells to NK cell-mediated cytotoxicity was induced by enforced expression of EHZF in the cervical carcinoma cell line HeLa and in the B lymphoblastoid cell line IM9. Preincubation of transfected cells with HLA class I Ag-specific mAb restored target cell susceptibility to NK cell-mediated lysis, indicating a specific role for HLA class I Ag up-regulation in the NK resistance induced by EHZF. A potential clinical significance of these findings is further suggested by the inverse correlation between EHZF and MHC class I expression levels, and autologous NK susceptibility of freshly explanted multiple myeloma cells. PMID:19342626

SMAD4 impedes the conversion of NK cells into ILC1-like cells by curtailing non-canonical TGF-β signaling.

PubMed

Cortez, Victor S; Ulland, Tyler K; Cervantes-Barragan, Luisa; Bando, Jennifer K; Robinette, Michelle L; Wang, Qianli; White, Andrew J; Gilfillan, Susan; Cella, Marina; Colonna, Marco

2017-09-01

Among the features that distinguish type 1 innate lymphoid cells (ILC1s) from natural killer (NK) cells is a gene signature indicative of 'imprinting' by cytokines of the TGF-β family. We studied mice in which ILC1s and NK cells lacked SMAD4, a signal transducer that facilitates the canonical signaling pathway common to all cytokines of the TGF-β family. While SMAD4 deficiency did not affect ILC1 differentiation, NK cells unexpectedly acquired an ILC1-like gene signature and were unable to control tumor metastasis or viral infection. Mechanistically, SMAD4 restrained non-canonical TGF-β signaling mediated by the cytokine receptor TGFβR1 in NK cells. NK cells from a SMAD4-deficient person affected by polyposis were also hyper-responsive to TGF-β. These results identify SMAD4 as a previously unknown regulator that restricts non-canonical TGF-β signaling in NK cells.

SMAD4 impedes the conversion of NK cells into ILC1-like cells by curtailing non-canonical TGF-β signaling

PubMed Central

Cortez, Victor S; Ulland, Tyler K; Cervantes-Barragan, Luisa; Bando, Jennifer K; Robinette, Michelle L; Wang, Qianli; White, Andrew J; Gilfillan, Susan; Cella, Marina; Colonna, Marco

2017-01-01

Among the features that distinguish type 1 innate lymphoid cells (ILC1s) from natural killer (NK) cells is a gene signature indicative of ‘imprinting’ by cytokines of the TGF-β family. We examined mice in which ILC1s and NK cells lacked SMAD4, a signal transducer that facilitates the canonical signaling pathway common to all cytokines of the TGF-β family. While SMAD4 deficiency did not affect ILC1 differentiation, NK cells unexpectedly acquired an ILC1-like gene signature and were unable to control tumor metastasis or viral infection. Mechanistically, SMAD4 restrained non-canonical TGF-β signaling mediated by the cytokine receptor TGF-βR1 in NK cells. NK cells from a SMAD4-deficient person affected by polyposis were also hyper-responsive to TGF-β. These results identify SMAD4 as a previously unknown regulator that restricts non-canonical TGF-β signaling in NK cells. PMID:28759002

Monocytes Play an IL-12-Dependent Crucial Role in Driving Cord Blood NK Cells to Produce IFN-g in Response to Trypanosoma cruzi

PubMed Central

Guilmot, Aline; Bosse, Julie; Carlier, Yves; Truyens, Carine

2013-01-01

We previously reported that foetuses congenitally infected with Trypanosoma cruzi, the agent of Chagas disease, mount an adult-like parasite-specific CD8+ T-cell response, producing IFN-g, and present an altered NK cell phenotype, possibly reflecting a post-activation state supported by the ability of the parasite to trigger IFN-g synthesis by NK cells in vitro. We here extended our knowledge on NK cell activation by the parasite. We compared the ability of T. cruzi to activate cord blood and adult NK cells from healthy individuals. Twenty-four hours co-culture of cord blood mononuclear cells with T. cruzi trypomastigotes and IL-15 induced high accumulation of IFN-g transcripts and IFN-g release. TNF-a, but not IL-10, was also produced. This was associated with up-regulation of CD69 and CD54, and down-regulation of CD62L on NK cells. The CD56bright NK cell subset was the major IFN-g responding subset (up to 70% IFN-g-positive cells), while CD56dim NK cells produced IFN-g to a lesser extent. The response points to a synergy between parasites and IL-15. The neonatal response, observed in all newborns, remained however slightly inferior to that of adults. Activation of IL-15-sensitized cord blood NK cells by the parasite required contacts with live/intact parasites. In addition, it depended on the engagement of TLR-2 and 4 and involved IL-12 and cross-talk with monocytes but not with myeloid dendritic cells, as shown by the use of neutralizing antibodies and cell depletion. This work highlights the ability of T. cruzi to trigger a robust IFN-g response by IL-15-sensitized human neonatal NK cells and the important role of monocytes in it, which might perhaps partially compensate for the neonatal defects of DCs. It suggests that monocyte- and IL-12- dependent IFN-g release by NK cells is a potentially important innate immune response pathway allowing T. cruzi to favour a type 1 immune response in neonates. PMID:23819002

Acetylcholine-producing NK cells attenuate CNS inflammation via modulation of infiltrating monocytes/macrophages.

PubMed

Jiang, Wei; Li, Daojing; Han, Ranran; Zhang, Chao; Jin, Wei-Na; Wood, Kristofer; Liu, Qiang; Shi, Fu-Dong; Hao, Junwei

2017-07-25

The nonneural cholinergic system of immune cells is pivotal for the maintenance of immunological homeostasis. Here we demonstrate the expression of choline acetyltransferase (ChAT) and cholinergic enzymes in murine natural killer (NK) cells. The capacity for acetylcholine synthesis by NK cells increased markedly under inflammatory conditions such as experimental autoimmune encephalomyelitis (EAE), in which ChAT expression escalated along with the maturation of NK cells. ChAT + and ChAT - NK cells displayed distinctive features in terms of cytotoxicity and chemokine/cytokine production. Transfer of ChAT + NK cells into the cerebral ventricles of CX3CR1 -/- mice reduced brain and spinal cord damage after EAE induction, and decreased the numbers of CNS-infiltrating CCR2 + Ly6C hi monocytes. ChAT + NK cells killed CCR2 + Ly6C hi monocytes directly via the disruption of tolerance and inhibited the production of proinflammatory cytokines. Interestingly, ChAT + NK cells and CCR2 + Ly6C hi monocytes formed immune synapses; moreover, the impact of ChAT + NK cells was mediated by α7-nicotinic acetylcholine receptors. Finally, the NK cell cholinergic system up-regulated in response to autoimmune activation in multiple sclerosis, perhaps reflecting the severity of disease. Therefore, this study extends our understanding of the nonneural cholinergic system and the protective immune effect of acetylcholine-producing NK cells in autoimmune diseases.

Regulation of bacteria population behaviors by AI-2 "consumer cells" and "supplier cells".

PubMed

Quan, Yufen; Meng, Fankang; Ma, Xinyu; Song, Xinhao; Liu, Xiao; Gao, Weixia; Dang, Yulei; Meng, Yao; Cao, Mingfeng; Song, Cunjiang

2017-09-19

Autoinducer-2 (AI-2) is a universal signal molecule and enables an individual bacteria to communicate with each other and ultimately control behaviors of the population. Harnessing the character of AI-2, two kinds of AI-2 "controller cells" ("consumer cells" and "supplier cells") were designed to "reprogram" the behaviors of entire population. For the consumer cells, genes associated with the uptake and processing of AI-2, which includes LsrACDB, LsrFG, LsrK, were overexpressed in varying combinations. Four consumer cell strains were constructed: Escherichia coli MG1655 pLsrACDB (NK-C1), MG1655 pLsrACDBK (NK-C2), MG1655 pLsrACDBFG (NK-C3) and MG1655 pLsrACDBFGK (NK-C4). The key enzymes responsible for production of AI-2, LuxS and Mtn, were also overexpressed, yielding strains MG1655 pLuxS (NK-SU1), and MG1655 pLuxS-Mtn (NK-SU2). All the consumer cells could decrease the environmental AI-2 concentration. NK-C2 and NK-C4 were most effective in AI-2 uptake and inhibited biofilm formation. While suppliers can increase the environmental AI-2 concentration and NK-SU2 was most effective in supplying AI-2 and facilitated biofilm formation. Further, reporter strain, MG1655 pLGFP was constructed. The expression of green fluorescent protein (GFP) in reporter cells was initiated and guided by AI-2. Mixture of consumer cells and reporter cells suggest that consumer cells can decrease the AI-2 concentration. And the supplier cells were co-cultured with reporter cells, indicating that supplier cells can provide more AI-2 compared to the control. The consumer cells and supplier cells could be used to regulate environmental AI-2 concentration and the biofilm formation. They can also modulate the AI-2 concentration when they were co-cultured with reporter cells. It can be envisioned that this system will become useful tools in synthetic biology and researching new antimicrobials.

Amino acid-dependent cMyc expression is essential for NK cell metabolic and functional responses in mice.

PubMed

Loftus, Róisín M; Assmann, Nadine; Kedia-Mehta, Nidhi; O'Brien, Katie L; Garcia, Arianne; Gillespie, Conor; Hukelmann, Jens L; Oefner, Peter J; Lamond, Angus I; Gardiner, Clair M; Dettmer, Katja; Cantrell, Doreen A; Sinclair, Linda V; Finlay, David K

2018-06-14

Natural killer (NK) cells are lymphocytes with important anti-tumour functions. Cytokine activation of NK cell glycolysis and oxidative phosphorylation (OXPHOS) are essential for robust NK cell responses. However, the mechanisms leading to this metabolic phenotype are unclear. Here we show that the transcription factor cMyc is essential for IL-2/IL-12-induced metabolic and functional responses in mice. cMyc protein levels are acutely regulated by amino acids; cMyc protein is lost rapidly when glutamine is withdrawn or when system L-amino acid transport is blocked. We identify SLC7A5 as the predominant system L-amino acid transporter in activated NK cells. Unlike other lymphocyte subsets, glutaminolysis and the tricarboxylic acid cycle do not sustain OXPHOS in activated NK cells. Glutamine withdrawal, but not the inhibition of glutaminolysis, results in the loss of cMyc protein, reduced cell growth and impaired NK cell responses. These data identify an essential role for amino acid-controlled cMyc for NK cell metabolism and function.

Innate NKTγδ and NKTαβ cells exert similar functions and compete for a thymic niche.

PubMed

Pereira, Pablo; Boucontet, Laurent

2012-05-01

The transcriptional regulator promyelocytic leukemia zinc finger (PLZF) is highly expressed during the differentiation of natural killer T (NKT) cells and is essential for the acquisition of their effector/memory innate-like phenotype. Staining with anti-PLZF and anti-NK1.1 Abs allows the definition of two subsets of NKTαβ and NKTγδ thymocytes that differ phenotypically and functionally: a PLZF(+) NK1.1(-) subset composed of mostly quiescent cells that secrete more IL-4 than IFN-γ upon activation and a PLZF(+/-) NK1.1(+) subset that expresses CD127, NK1.1, and other NK-cell markers, secrete more IFN-γ than IL-4 upon activation and contains a sizable fraction of dividing cells. The size of the NK1.1(+) population is very tightly regulated and NK1.1(+) αβ and γδ thymocytes compete for a thymic niche. Furthermore, the relative representation of the PLZF(+) and NK1.1(+) subsets varies in a strain-specific manner with C57BL/6 (B6) mice containing more NK1.1(+) cells and (B6 × DBA/2)F1 (B6D2F1) mice more PLZF(+) cells. Consequently, activation of NKT cells in vivo is expected to result in higher levels of IL-4 secreted in B6D2F1 mice than in B6 mice. Consistent with this possibility, B6D2F1 mice, when compared with B6 mice, contain more "innate" CD8(+) thymocytes, the generation of which depends on IL-4 secreted by NKT cells. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulatory role of NKG2D+ NK cells in intestinal lamina propria by secreting double-edged Th1 cytokines in ulcerative colitis

PubMed Central

Lin, Xue; Chang, Ying; Liu, Jing; Zhou, Rui; Nie, Jia-Yan; Dong, Wei-Guo; Zhao, Qiu; Li, Jin

2017-01-01

The role of intestinal lamina propria (LP) NKG2D+ NK cells is unclear in regulating Th1/Th2 balance in ulcerative colitis (UC). In this study, we investigated the frequency of LP NKG2D+ NK cells in DSS-induced colitis model and intestinal mucosal samples of UC patients, as well as the secretion of Th1/Th2/Th17 cytokines in NK cell lines after MICA stimulation. The role of Th1 cytokines in UC was validated by bioinformatics analysis. We found that DSS-induced colitis in mice was characterized by a Th2-mediated process. In acute phrase, the frequency of LP NKG2D+ lymphocytes increased significantly and decreased in remission, while the frequency of LP NKG2D+ NK cells decreased significantly in acute phase and increased in remission. No obvious change was found in the frequency of total LP NK cells. Similarly, severe UC patients had a higher expression of mucosal NKG2D and a lower number of NKG2D+ NK cells than mild to moderate UC. In NK cell lines, the MICA stimulation could induce a predominant secretion of Th1 cytokines (TNF, IFN-γ). Furthermore, in bioinformatics analysis, mucosal Th1 cytokine of TNF, showed a double-edged role in UC when compared to the Th1-mediated disease of Crohn's colitis. In conclusion, LP NKG2D+ NK cells partially played a regulatory role in UC through secreting Th1 cytokines to regulate the Th2-predominant Th1/Th2 imbalance, despite of the concomitant pro-inflammatory effects of Th1 cytokines. PMID:29228739

Histaminergic regulation of interferon-gamma (IFN-γ) production by human natural killer (NK) cells

PubMed Central

ASEA, A; HANSSON, M; CZERKINSKY, C; HOUZE, T; HERMODSSON, S; STRANNEGÅRD, Ö; HELLSTRAND, K

1996-01-01

Monocytes, recovered from human peripheral blood by counter-current centrifugal elutriation, effectively inhibit the production of IFN-γ by CD3−/56+ NK cells in response to IL-2. This study aimed at defining the nature of the inhibitory signal, particularly the importance of monocyte-derived reactive metabolites of oxygen. It was found that monocytes recovered from patients with chronic granulomatous disease (CGD), a condition characterized by deficient NADPH-oxidase activity of phagocytes, did not inhibit IFN-γ production by NK cells. Further, catalase, a scavenger of hydrogen peroxide, completely reversed the inhibitory signal, whereas scavengers of the superoxide anion, hypohalous acids, the hydroxyl radical, or nitric oxide synthesis inhibitors such as L-NMMA were ineffective. Inhibition of IFN-γ production was operating on a pre-translational level, as indicated by the inability of enriched NK cells to accumulate IFN-γ mRNA in the presence of elutriated monocytes. Hydrogen peroxide, at micromolar concentrations, reconstituted the inhibition of IFN-γ production when added to enriched NK cells. Histamine, a biogenic amine which inhibits the generation of reactive oxygen metabolites in monocytes, abrogated the inhibition of IFN-γ production in NK cells; by this mechanism, histamine strongly synergized with IL-2 to induce IFN-γ in mixtures of NK cells and monocytes. The synergizing effect of histamine was specifically mediated by H2-type histamine receptors. We conclude that: (i) the induction of IFN-γ mRNA in NK cells is effectively down-regulated by products of the oxidative metabolism of monocytes; and (ii) histamine effectively enhances IFN-γ production by preventing monocyte-induced oxidative damage to NK cells. PMID:8706348

Histaminergic regulation of interferon-gamma (IFN-gamma) production by human natural killer (NK) cells.

PubMed

Asea, A; Hansson, M; Czerkinsky, C; Houze, T; Hermodsson, S; Strannegård, O; Hellstrand, K

1996-08-01

Monocytes, recovered from human peripheral blood by counter-current centrifugal elutriation, effectively inhibit the production of IFN-gamma by CD3-/56+ NK cells in response to IL-2. This study aimed at defining the nature of the inhibitory signal, particularly the importance of monocyte-derived reactive metabolites of oxygen. It was found that monocytes recovered from patients with chronic granulomatous disease (CGD), a condition characterized by deficient NADPH-oxidase activity of phagocytes, did not inhibit IFN-gamma production by NK cells. Further, catalase, a scavenger of hydrogen peroxide, completely reversed the inhibitory signal whereas scavengers of the superoxide anion, hypohalous acids, the hydroxyl radical, or nitric oxide synthesis inhibitors such as L-NMMA were ineffective. Inhibition of IFN-gamma production was operating on a pretranslational level, as indicated by the inability of enriched NK cells to accumulate IFN-gamma mRNA in the presence of elutriated monocytes. Hydrogen peroxide, at micromolar concentrations, reconstituted the inhibition of IFN-gamma production when added to enriched NK cells. Histamine, a biogenic amine which inhibits the generation of reactive oxygen metabolites in monocytes, abrogated the inhibition of IFN-gamma production in NK cells; by this mechanism, histamine strongly synergized with IL-2 to induce IFN-gamma in mixtures of NK cells and monocytes. The synergizing effect of histamine was specifically mediated by H2-type histamine receptors. We conclude that: (i) the induction of IFN-gamma mRNA in NK cells is effectively down-regulated by products of the oxidative metabolism of monocytes; and (ii) histamine effectively enhances IFN-gamma production by preventing monocyte-induced oxidative damage to NK cells.

Critical role for the chemokine receptor CXCR6 in homeostasis and activation of CD1d-restricted NKT cells.

PubMed

Germanov, Elitza; Veinotte, Linnea; Cullen, Robyn; Chamberlain, Erin; Butcher, Eugene C; Johnston, Brent

2008-07-01

NK T (NKT) cells play important roles in the regulation of diverse immune responses. However, little is known about the mechanisms that regulate homeostasis and activation of these cells. Thymic NKT cells up-regulated the chemokine receptor CXCR6 following positive selection and migrated toward CXCL16 in vitro. However, CXCR6 was not essential for thymic development or maturation. In contrast, liver and lung NKT cells were depleted in CXCR6+/- and CXCR6-/- mice. The reduction in liver and lung NKT cells coincided with an increase in bone marrow NKT cells, suggesting a redistribution of NKT cells in CXCR6-/- animals. In wild-type mice, CXCL16 neutralization reduced accumulation of mature NK1.1+, but not immature NK1.1- NKT cell recent thymic emigrants in the liver. Given that thymic NKT cells are preferentially exported as NK1.1- cells, this suggests an additional role for CXCR6/CXCL16 in maturation or survival of immature liver NKT cells. CXCL16 blockade did not deplete resident NK1.1+ NKT cells, indicating that CXCR6/CXCL16 are not required to retain mature NKT cells in the liver. Cytokine production by liver and spleen NKT cells was impaired in CXCR6-/- mice following in vivo stimulation with alpha-galactosylceramide, implicating a novel role for CXCR6 in NKT cell activation. Reduced IFN-gamma production was not due to an intrinsic defect as production was normal following PMA and ionomycin stimulation. Preformed transcripts for IL-4, but not IFN-gamma, were reduced in CXCR6-/- liver NKT cells. These data identify critical roles for CXCR6/CXCL16 in NKT cell activation and the regulation of NKT cell homeostasis.

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

PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells.

PubMed

Miranda, Dante; Jara, Claudia; Ibañez, Jorge; Ahumada, Viviana; Acuña-Castillo, Claudio; Martin, Adrian; Córdova, Alexandra; Montoya, Margarita

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.

Human liver-resident CD56(bright)/CD16(neg) NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways.

PubMed

Hudspeth, Kelly; Donadon, Matteo; Cimino, Matteo; Pontarini, Elena; Tentorio, Paolo; Preti, Max; Hong, Michelle; Bertoletti, Antonio; Bicciato, Silvio; Invernizzi, Pietro; Lugli, Enrico; Torzilli, Guido; Gershwin, M Eric; Mavilio, Domenico

2016-01-01

The liver-specific natural killer (NK) cell population is critical for local innate immune responses, but the mechanisms that lead to their selective homing and the definition of their functionally relevance remain enigmatic. We took advantage of the availability of healthy human liver to rigorously define the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts. Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56(bright) lr-NK cells that localize within hepatic sinusoids. CD56(bright) lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally critical as it determines selective migration in response to the chemotactic stimuli exerted by CCL3, CCL5 and CXCL16. Here, we also show that hepatic sinusoids express CCL3(pos) Kupffer cells, CXCL16(pos) endothelial cells and CCL5(pos) T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a unique tissue niche for lr-CD56(bright) NK cells that constitutively express CCR5 and CXCR6. CD56(bright) lr-NK cells co-exist with CD56(dim) conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56(dim) c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX3CR1. Our findings disclosing the phenotypic and functional differences between lr-Nk cells and c-NK cells are critical to distinguish liver-specific innate immune responses. Hence, any therapeutic attempts at modifying the large population of CD56(bright) lr-NK cells will require modification of hepatic CCR5 and CXCR6. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human liver-resident CD56bright/CD16neg NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways

PubMed Central

Hudspeth, Kelly; Donadon, Matteo; Cimino, Matteo; Pontarini, Elena; Tentorio, Paolo; Preti, Max; Hong, Michelle; Bertoletti, Antonio; Bicciato, Silvio; Invernizzi, Pietro; Lugli, Enrico; Torzilli, Guido; Gershwin, M. Eric; Mavilio, Domenico

2015-01-01

Rationale The liver-specific natural killer (NK) cell population is critical for local innate immune responses, but the mechanisms that lead to their selective homing and the definition of their functionally relevance remain enigmatic. Objectives We took advantage of the availability of healthy human liver to rigorously define the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts. Findings Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56bright lr-NK cells that localize within hepatic sinusoids. Further, CD56bright lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally critical as it determines selective migration in response to the chemotactic stimuli exerted by CCL3, CCL5 and CXCL16. In addition, hepatic sinusoids express CCL3pos Kupffer cells, CXCL16pos endothelial cells and CCL5pos T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a tissue niche for lr-CD56bright NK cells that constitutively express CCR5 and CXCR6. CD56bright lr-NK cells co-exist with CD56dim conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56dim c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX3CR1. Conclusion Our findings disclosing the phenotypic and functional differences between lr-Nk cells and c-NK cells are critical to distinguish liver-specific innate immune responses. Hence, any therapeutic attempts at modifying the large population of CD56bright lr-NK cells will require modification of hepatic CCR5 and CXCR6. PMID:26330348

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

PubMed Central

LAPTEVA, NATALIA; DURETT, APRIL G.; SUN, JIALI; ROLLINS, LISA A.; HUYE, LESLIE L.; FANG, JIAN; DANDEKAR, VARADA; MEI, ZHUYONG; JACKSON, KIMBERLEY; VERA, JUAN; ANDO, JUN; NGO, MINHTRAN C.; COUSTAN-SMITH, ELAINE; CAMPANA, DARIO; SZMANIA, SUSANN; GARG, TARUN; MORENO-BOST, AMBERLY; VANRHEE, FRITS; GEE, ADRIAN P.; ROONEY, CLIONA M.

2016-01-01

Background aims Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. Methods We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Results Using this system we produced up to 19 × 109 functional NK cells from unseparated apheresis products, starting with 15 × 107 CD3− CD56+ NK cells, within 8–10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8+ T cells within the NK cultures. However, these CD3+ T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. Conclusions We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy. PMID:22900959

Natural killer cells: In health and disease.

PubMed

Mandal, Arundhati; Viswanathan, Chandra

2015-06-01

Natural killer (NK) cells constitute our bodies' frontline defense system, guarding against tumors and launching attacks against infections. The activities of NK cells are regulated by the interaction of various receptors expressed on their surfaces with cell surface ligands. While the role of NK cells in controlling tumor activity is relatively clear, the fact that they are also linked to various other disease conditions is now being highlighted. Here, we present an overview of the role of NK cells during normal body state as well as under diseased state. We discuss the possible utilization of these powerful cells as immunotherapeutic agents in combating diseases such as asthma, autoimmune diseases, and HIV-AIDS. This review also outlines current challenges in NK cell therapy. Copyright © 2015. Published by Elsevier B.V.

The anti-tumor role of NK cells in vivo pre-activated and re-stimulated by interleukins in acute lymphoblastic leukemia

PubMed Central

Jin, Fengyan; Lin, Hai; Gao, Sujun; Hu, Zheng; Zuo, Song; Sun, Liguang; Jin, Chunhui; Li, Wei; Yang, Yanping

2016-01-01

Although natural killer cells (NK cells) were traditionally classified as members of the innate immune system, NK cells have recently been found also to be an important player in the adaptive immune systems. In this context, in vitro activation of NK cells by cytokines leads to generation of NK cells with memory-like properties characterized by increased interferon-γ (IFNγ) production. However, it remains to be defined whether these memory-like NK cells exist in vivo after cytokine activation. Furthermore, it is also unclear whether such memory-like NK cells induced in vivo by cytokines could have effective anti-leukemia response. To address these issues, we used an in vivo pre-activation and re-stimulation system that was able to produce NK cells with increased IFNγ secretion. It was found that after in vivo pre-activation and re-stimulation with interleukins (ILs), NK cells retained a state to produce increased amount of IFNγ. Of note, whereas this intrinsic capacity of enhanced IFNγ production after in vivo IL pre-activation and re-stimulation could be transferred to the next generation of NK cells and was associated with prolonged survival of the mice with acute lymphoid leukemia. Moreover, the anti-leukemia activity of these memory-like NK cells was associated with IFNγ production and up-regulation of NK cells activation receptor-NK Group 2 member D (NKG2D). Together, these findings argue strongly that in vivo IL pre-activation and re-stimulation is capable to induce memory-like NK cells as observed previously in vitro, which are effective against acute lymphoblastic leukemia, likely via NKG2D-dependent IFNγ production, in intact animals. PMID:27816971

The anti-tumor role of NK cells in vivo pre-activated and re-stimulated by interleukins in acute lymphoblastic leukemia.

PubMed

Jin, Fengyan; Lin, Hai; Gao, Sujun; Hu, Zheng; Zuo, Song; Sun, Liguang; Jin, Chunhui; Li, Wei; Yang, Yanping

2016-11-29

Although natural killer cells (NK cells) were traditionally classified as members of the innate immune system, NK cells have recently been found also to be an important player in the adaptive immune systems. In this context, in vitro activation of NK cells by cytokines leads to generation of NK cells with memory-like properties characterized by increased interferon-γ (IFNγ) production. However, it remains to be defined whether these memory-like NK cells exist in vivo after cytokine activation. Furthermore, it is also unclear whether such memory-like NK cells induced in vivo by cytokines could have effective anti-leukemia response. To address these issues, we used an in vivo pre-activation and re-stimulation system that was able to produce NK cells with increased IFNγ secretion. It was found that after in vivo pre-activation and re-stimulation with interleukins (ILs), NK cells retained a state to produce increased amount of IFNγ. Of note, whereas this intrinsic capacity of enhanced IFNγ production after in vivo IL pre-activation and re-stimulation could be transferred to the next generation of NK cells and was associated with prolonged survival of the mice with acute lymphoid leukemia. Moreover, the anti-leukemia activity of these memory-like NK cells was associated with IFNγ production and up-regulation of NK cells activation receptor-NK Group 2 member D (NKG2D). Together, these findings argue strongly that in vivo IL pre-activation and re-stimulation is capable to induce memory-like NK cells as observed previously in vitro, which are effective against acute lymphoblastic leukemia, likely via NKG2D-dependent IFNγ production, in intact animals.

The Transcription Factor ZNF683/HOBIT Regulates Human NK-Cell Development

PubMed Central

Post, Mirte; Cuapio, Angelica; Osl, Markus; Lehmann, Dorit; Resch, Ulrike; Davies, David M.; Bilban, Martin; Schlechta, Bernhard; Eppel, Wolfgang; Nathwani, Amit; Stoiber, Dagmar; Spanholtz, Jan; Casanova, Emilio; Hofer, Erhard

2017-01-01

We identified ZNF683/HOBIT as the most highly upregulated transcription factor gene during ex vivo differentiation of human CD34+ cord blood progenitor cells to CD56+ natural killer (NK) cells. ZNF683/HOBIT mRNA was preferentially expressed in NK cells compared to other human peripheral blood lymphocytes and monocytes. During ex vivo differentiation, ZNF683/HOBIT mRNA started to increase shortly after addition of IL-15 and further accumulated in parallel to the generation of CD56+ NK cells. shRNA-mediated knockdown of ZNF683/HOBIT resulted in a substantial reduction of CD56−CD14− NK-cell progenitors and the following generation of CD56+ NK cells was largely abrogated. The few CD56+ NK cells, which escaped the developmental inhibition in the ZNF683/HOBIT knockdown cultures, displayed normal levels of NKG2A and KIR receptors. Functional analyses of these cells showed no differences in degranulation capacity from control cultures. However, the proportion of IFN-γ-producing cells appeared to be increased upon ZNF683/HOBIT knockdown. These results indicate a key role of ZNF683/HOBIT for the differentiation of the human NK-cell lineage and further suggest a potential negative control on IFN-γ production in more mature human NK cells. PMID:28555134

The Transcription Factor ZNF683/HOBIT Regulates Human NK-Cell Development.

PubMed

Post, Mirte; Cuapio, Angelica; Osl, Markus; Lehmann, Dorit; Resch, Ulrike; Davies, David M; Bilban, Martin; Schlechta, Bernhard; Eppel, Wolfgang; Nathwani, Amit; Stoiber, Dagmar; Spanholtz, Jan; Casanova, Emilio; Hofer, Erhard

2017-01-01

We identified ZNF683/HOBIT as the most highly upregulated transcription factor gene during ex vivo differentiation of human CD34 + cord blood progenitor cells to CD56 + natural killer (NK) cells. ZNF683/HOBIT mRNA was preferentially expressed in NK cells compared to other human peripheral blood lymphocytes and monocytes. During ex vivo differentiation, ZNF683/HOBIT mRNA started to increase shortly after addition of IL-15 and further accumulated in parallel to the generation of CD56 + NK cells. shRNA-mediated knockdown of ZNF683/HOBIT resulted in a substantial reduction of CD56 - CD14 - NK-cell progenitors and the following generation of CD56 + NK cells was largely abrogated. The few CD56 + NK cells, which escaped the developmental inhibition in the ZNF683/HOBIT knockdown cultures, displayed normal levels of NKG2A and KIR receptors. Functional analyses of these cells showed no differences in degranulation capacity from control cultures. However, the proportion of IFN-γ-producing cells appeared to be increased upon ZNF683/HOBIT knockdown. These results indicate a key role of ZNF683/HOBIT for the differentiation of the human NK-cell lineage and further suggest a potential negative control on IFN-γ production in more mature human NK cells.

Effect of anti-IL-15 administration on T cell and NK cell homeostasis in rhesus macaques

PubMed Central

DeGottardi, Maren Q.; Okoye, Afam A.; Vaidya, Mukta; Talla, Aarthi; Konfe, Audrie L.; Reyes, Matthew D.; Clock, Joseph A.; Duell, Derick M.; Legasse, Alfred W.; Sabnis, Amit; Park, Byung S.; Axthelm, Michael K.; Estes, Jacob D.; Reinmann, Keith A.; Sekaly, Rafick-Pierre; Picker, Louis J.

2016-01-01

IL-15 has been implicated as a key regulator of T and NK cell homeostasis in multiple systems; however, its specific role in maintaining peripheral T and NK cell populations relative to other gamma-chain (γc) cytokines has not been fully defined in primates. Here, we address this question by determining the effect of IL-15 inhibition with a rhesusized, anti-IL-15 mAb on T and NK cell dynamics in rhesus macaques. Strikingly, anti-IL-15 treatment resulted in rapid depletion of NK cells, and both CD4+ and CD8+ effector memory T cells (TEM) in blood and tissues, with little to no effect on naïve or central memory T cells. Importantly, whereas depletion of NK cells was nearly complete and maintained as long as anti-IL-15 treatment was given, TEM depletion was countered by the onset of massive TEM proliferation, which almost completely restored circulating TEM numbers. Tissue TEM, however, remained significantly reduced, and most TEM maintained very high turnover throughout anti-IL-15 treatment. In the presence of IL-15 inhibition, TEM became increasingly more sensitive to IL-7 stimulation in vivo, and transcriptional analysis of TEM in IL-15-inhibited monkeys revealed engagement of the JAK/STAT signaling pathway, suggesting alternative γc cytokine signaling may support TEM homeostasis in the absence of IL-15. Thus, IL-15 plays a major role in peripheral maintenance of NK cells and TEM. However, whereas most NK cell populations collapse in the absence of IL-15, TEM can be maintained in the face of IL-15 inhibition by the activity of other homeostatic regulators, most likely IL-7. PMID:27430715

Activated human primary NK cells efficiently kill colorectal cancer cells in 3D spheroid cultures irrespectively of the level of PD-L1 expression.

PubMed

Lanuza, Pilar M; Vigueras, Alan; Olivan, Sara; Prats, Anne C; Costas, Santiago; Llamazares, Guillermo; Sanchez-Martinez, Diego; Ayuso, José María; Fernandez, Luis; Ochoa, Ignacio; Pardo, Julián

2018-01-01

Haploidentical Natural Killer (NK) cells have been shown as an effective and safe alternative for the treatment of haematological malignancies with poor prognosis for which traditional therapies are ineffective. In contrast to haematological cancer cells, that mainly grow as single suspension cells, solid carcinomas are characterised by a tridimensional (3D) architecture that provide specific surviving advantages and resistance against chemo- and radiotherapy. However, little is known about the impact of 3D growth on solid cancer immunotherapy especially adoptive NK cell transfer. We have recently developed a protocol to activate ex vivo human primary NK cells using B lymphoblastic cell lines, which generates NK cells able to overcome chemoresistance in haematological cancer cells. Here we have analysed the activity of these allogeneic NK cells against colorectal (CRC) human cell lines growing in 3D spheroid culture and correlated with the expression of some of the main ligands regulating NK cell activity. Our results indicate that activated NK cells efficiently kill colorectal tumour cell spheroids in both 2D and 3D cultures. Notably, although 3D CRC cell cultures favoured the expression of the inhibitory immune checkpoint PD-L1, it did not correlate with increased resistance to NK cells. Finally, we have analysed in detail the infiltration of NK cells in 3D spheroids by microscopy and found that at low NK cell density, cell death is not observed although NK cells are able to infiltrate into the spheroid. In contrast, higher densities promote tumoural cell death before infiltration can be detected. These findings show that highly dense activated human primary NK cells efficiently kill colorectal carcinoma cells growing in 3D cultures independently of PD-L1 expression and suggest that the use of allogeneic activated NK cells could be beneficial for the treatment of colorectal carcinoma.

Expression patterns of lectin-like natural killer receptors, inhibitory CD94/NKG2A, and activating CD94/NKG2C on decidual CD56bright natural killer cells differ from those on peripheral CD56dim natural killer cells.

PubMed

Kusumi, Maki; Yamashita, Takahiro; Fujii, Tomoyuki; Nagamatsu, Takeshi; Kozuma, Shiro; Taketani, Yuji

2006-06-01

The balance of inhibitory and activating natural killer (NK) receptors on maternal decidual NK cells, most of which are CD56bright, is thought to be crucial for the proper growth of trophoblasts in placenta. A lectin-like NK receptor, CD94/NKG2, is the receptor for human leukocyte antigen (HLA)-E, which is expressed on trophoblasts. To clarify the mechanism regulating the activity of decidual NK cells during pregnancy, we investigated the expression patterns of inhibitory NK receptor, CD94/NKG2A, and activating receptor, CD94/NKG2C, on decidual NK cells in an early stage of normal pregnancy and compared them with those on peripheral NK cells, most of which are CD56dim. The rate of NKG2A-positive cells was significantly higher for decidual CD56bright NK cells than for peripheral CD56dim NK cells, but the rates of NKG2C-positive cells were comparable between the two cell types. Interestingly, peripheral CD56dim NK cells reciprocally expressed inhibitory NKG2A and activating NKG2C, but decidual CD56bright NK cells that expressed activating NKG2C simultaneously expressed inhibitory NKG2A. The co-expression of inhibitory and activating NKG2 receptors may fine-tune the immunoregulatory functions of the decidual NK cells to control the trophoblast invasion in constructing placenta.

Application of tissue-specific NK and NKT cell activity for tumor immunotherapy

PubMed Central

Subleski, Jeff J.; Wiltrout, Robert H.; Weiss, Jonathan M.

2009-01-01

Natural killer (NK) and NKT cells are a first line of defense against pathogens and transformed cells. However, dysregulation of their function can lead to autoimmune disease. A better understanding of the mechanisms controlling NK and NKT effector function should lead to the development of improved strategies for the treatment of many diseases. The site in which NK and NKT cells reside should be taken into account, because accumulating evidence suggests that the tissue microenvironment strongly influences their function. In this regard, the liver represents a unique immunologic organ in which the balance between the need for tolerance and the ability to respond rapidly to pathogens and tissue injury is tightly regulated. NK cells in the liver have augmented cytolytic activity as compared to other organs, which is consistent with a role for liver-associated NK cells in being critical effector cells for inhibiting tumor metastasis in the liver. Several studies also suggest that hepatic NKT cells have different functions than those in other organs. Whereas splenic and thymic NKT cells have been shown to suppress diabetes development, facilitate the induction of systemic tolerance and are regulated by IL-4 and other Th2 cytokines, certain subsets of NKT cells in the liver are important sources of Th1 cytokines such as Interferon gamma, and are the primary mediators of anti-tumor responses. The unique properties and roles as critical effector cells make NK and NKT cells within the liver microenvironment attractive targets of immunotherapeutic approaches that have the goal of controlling tumor metastasis in the liver. PMID:19682859

Lidocaine Stimulates the Function of Natural Killer Cells in Different Experimental Settings.

PubMed

Cata, Juan P; Ramirez, Maria F; Velasquez, Jose F; Di, A I; Popat, Keyuri U; Gottumukkala, Vijaya; Black, Dahlia M; Lewis, Valerae O; Vauthey, Jean N

2017-09-01

One of the functions of natural killer (NK) cells is to eliminate cancer cells. The cytolytic activity of NK cells is tightly regulated by inhibitory and activation receptors located in the surface membrane. Lidocaine stimulates the function of NK cells at clinically relevant concentrations. It remains unknown whether this effect of lidocaine has an impact on the expression of surface receptors of NK cells, can uniformly stimulate across different cancer cell lines, and enhances the function of cells obtained during oncological surgery. NK cells from healthy donors and 43 patients who had undergone surgery for cancer were isolated. The function of NK cells was measured by lactate dehydrogenase release assay. NK cells were incubated with clinically relevant concentrations of lidocaine. By flow cytometry, we determined the impact of lidocaine on the expression of galactosylgalactosylxylosylprotein3-beta-glucuronosytranferase 1, marker of cell maturation (CD57), killer cell lectin like receptor A, inhibitory (NKG2A) receptors and killer cell lectin like receptor D, activation (NKG2D) receptors of NK cells. Differences in expression at p<0.05 were considered statistically significant. Lidocaine increased the expression of NKG2D receptors and stimulated the function of NK cells against ovarian, pancreatic and ovarian cancer cell lines. Lidocaine also increased the cytolytic activity of NK cells from patients who underwent oncological surgery, except for those who had orthopedic procedures. Lidocaine showed an important stimulatory activity on NK cells. Our findings suggest that lidocaine might be used perioperatively to minimize the impact of surgery on NK cells. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Proteomic comparison of 3D and 2D glioma models reveals increased HLA-E expression in 3D models is associated with resistance to NK cell-mediated cytotoxicity.

PubMed

He, Weiqi; Kuang, Yongqin; Xing, Xuemin; Simpson, Richard J; Huang, Haidong; Yang, Tao; Chen, Jingmin; Yang, Libin; Liu, Enyu; He, Weifeng; Gu, Jianwen

2014-05-02

Three-dimensional cell culture techniques can better reflect the in vivo characteristics of tumor cells compared with traditional monolayer cultures. Compared with their 2D counterparts, 3D-cultured tumor cells showed enhanced resistance to the cytotoxic T cell-mediated immune response. However, it remains unclear whether 3D-cultured tumor cells have an enhanced resistance to NK cell cytotoxicity. In this study, a total of 363 differentially expressed proteins were identified between the 2D- and 3D-cultured U251 cells by comparative proteomics, and an immune-associated protein-protein interaction (PPI) network based on these differential proteins was constructed by bioinformatics. Within the network, HLA-E, as a molecule for inhibiting NK cell activation, was significantly up-regulated in the 3D-cultured tumor cells. Then, we found that the 3D-cultured U251 cells exhibited potent resistance to NK cell cytotoxicity in vitro and were prone to tumor formation in vivo. The resistance of the 3D-cultured tumor cells to NK cell lysis was mediated by the HLA-E/NKG2A interaction because the administration of antibodies that block either HLA-E or NKG2A completely eliminated this resistance and significantly decreased tumor formation. Taken together, our findings indicate that HLA-E up-regulation in 3D-cultured cells may result in enhanced tumor resistance to NK cell-mediated immune response.

Coengagement of CD16 and CD94 receptors mediates secretion of chemokines and induces apoptotic death of naive natural killer cells.

PubMed

Jewett, Anahid; Cacalano, Nicholas A; Head, Christian; Teruel, Antonia

2006-04-01

Down-modulation of CD16 (FcgammaRIII) receptors and loss of natural killer (NK) cell function have been observed in oral cancer patients. However, neither the mechanisms nor the significance of the decrease in CD16 receptors have been fully understood. The cytotoxic activity and survival of NK cells are negatively regulated by antibodies directed against CD16 surface receptor. The addition of anti-CD94 antibody in combination with either F(ab')(2) fragment or intact anti-CD16 antibody to NK cells resulted in significant inhibition of NK cell cytotoxic function and induction of apoptosis in resting human peripheral blood NK cells. Addition of interleukin-2 to anti-CD16 and/or anti-CD94 antibody-treated NK cells significantly inhibited apoptosis and increased the function of NK cells. There was a significant increase in tumor necrosis factor-alpha (TNF-alpha) but not IFN-gamma secretion in NK cells treated either with anti-CD16 antibody alone or in combination with anti-CD94 antibodies. Consequently, the addition of anti-TNF-alpha antibody partially inhibited apoptosis of NK cells mediated by the combination of anti-CD94 and anti-CD16 antibodies. Increase in apoptotic death of NK cells also correlated with an increase in type 2 inflammatory cytokines and in the induction of chemokines. Thus, we conclude that binding of antibodies to CD16 and CD94 NK cell receptors induces death of the NK cells and signals for the release of chemokines.

Trichostatin A Sensitizes Hepatocellular Carcinoma Cells to Enhanced NK Cell-mediated Killing by Regulating Immune-related Genes.

PubMed

Shin, Sangsu; Kim, Miok; Lee, Seon-Jin; Park, Kang-Seo; Lee, Chang Hoon

2017-01-01

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. The ability of HCC to avoid immune detection is considered one of the main factors making it difficult to cure. Abnormal histone deacetylation is thought to be one of the mechanisms for HCC immune escape, making histone deacetylases (HDACs) attractive targets for HCC treatment. Here, we investigated the effect of trichostatin A (TSA), a highly potent HDAC inhibitor, on HCC (HepG2) gene expression and function. A genome wide-transcriptional microarray was used to identify genes regulated by TSA in HepG2 cells. Gene Ontology was used to identify pathways regulated by TSA, and these changes were confirmed by qPCR. The effect of TSA on natural killer (NK) cell-mediated killing of HCC cell lines were analyzed by both flow cytometry and LDH cytotoxicity assay. A study was also conducted in a Balb/c nude mice xenograft model to assess the anti-tumor activity of TSA. TSA regulated the transcription of numerous innate immunity & tumor antigen recognition-associated genes, such as ULBP1 and RAET1G, in HCC cells. In vivo, TSA reduced tumor cell growth in an NK cell-dependent manner. In vitro, TSA treatment of HepG2 cells rendered them more susceptible to NK cell-mediated killing while increasing the expression of NKGD2 ligands, including ULBP1/2/3 and MICA/B. TSA also induced direct killing of HCC cells by stimulating apoptosis. TSA likely increases killing of HCC cells indirectly by increasing NK cell-directed killing and directly by increasing apoptosis. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Trichostatin A Sensitizes Hepatocellular Carcinoma Cells to Enhanced NK Cell-mediated Killing by Regulating Immune-related Genes

PubMed Central

SHIN, SANGSU; KIM, MIOK; LEE, SEON-JIN; PARK, KANG-SEO

2017-01-01

Background/Aim: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. The ability of HCC to avoid immune detection is considered one of the main factors making it difficult to cure. Abnormal histone deacetylation is thought to be one of the mechanisms for HCC immune escape, making histone deacetylases (HDACs) attractive targets for HCC treatment. Here, we investigated the effect of trichostatin A (TSA), a highly potent HDAC inhibitor, on HCC (HepG2) gene expression and function. Materials and Methods: A genome wide-transcriptional microarray was used to identify genes regulated by TSA in HepG2 cells. Gene Ontology was used to identify pathways regulated by TSA, and these changes were confirmed by qPCR. The effect of TSA on natural killer (NK) cell-mediated killing of HCC cell lines were analyzed by both flow cytometry and LDH cytotoxicity assay. A study was also conducted in a Balb/c nude mice xenograft model to assess the anti-tumor activity of TSA. Results: TSA regulated the transcription of numerous innate immunity & tumor antigen recognition-associated genes, such as ULBP1 and RAET1G, in HCC cells. In vivo, TSA reduced tumor cell growth in an NK cell-dependent manner. In vitro, TSA treatment of HepG2 cells rendered them more susceptible to NK cell-mediated killing while increasing the expression of NKGD2 ligands, including ULBP1/2/3 and MICA/B. TSA also induced direct killing of HCC cells by stimulating apoptosis. Conclusion: TSA likely increases killing of HCC cells indirectly by increasing NK cell-directed killing and directly by increasing apoptosis. PMID:28871002

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

Genetic engineering of human NK cells to express CXCR2 improves migration to renal cell carcinoma.

PubMed

Kremer, Veronika; Ligtenberg, Maarten A; Zendehdel, Rosa; Seitz, Christina; Duivenvoorden, Annet; Wennerberg, Erik; Colón, Eugenia; Scherman-Plogell, Ann-Helén; Lundqvist, Andreas

2017-09-19

Adoptive natural killer (NK) cell transfer is being increasingly used as cancer treatment. However, clinical responses have so far been limited to patients with hematological malignancies. A potential limiting factor in patients with solid tumors is defective homing of the infused NK cells to the tumor site. Chemokines regulate the migration of leukocytes expressing corresponding chemokine receptors. Various solid tumors, including renal cell carcinoma (RCC), readily secrete ligands for the chemokine receptor CXCR2. We hypothesize that infusion of NK cells expressing high levels of the CXCR2 chemokine receptor will result in increased influx of the transferred NK cells into tumors, and improved clinical outcome in patients with cancer. Blood and tumor biopsies from 14 primary RCC patients were assessed by flow cytometry and chemokine analysis. Primary NK cells were transduced with human CXCR2 using a retroviral system. CXCR2 receptor functionality was determined by Calcium flux and NK cell migration was evaluated in transwell assays. We detected higher concentrations of CXCR2 ligands in tumors compared with plasma of RCC patients. In addition, CXCL5 levels correlated with the intratumoral infiltration of CXCR2-positive NK cells. However, tumor-infiltrating NK cells from RCC patients expressed lower CXCR2 compared with peripheral blood NK cells. Moreover, healthy donor NK cells rapidly lost their CXCR2 expression upon in vitro culture and expansion. Genetic modification of human primary NK cells to re-express CXCR2 improved their ability to specifically migrate along a chemokine gradient of recombinant CXCR2 ligands or RCC tumor supernatants compared with controls. The enhanced trafficking resulted in increased killing of target cells. In addition, while their functionality remained unchanged compared with control NK cells, CXCR2-transduced NK cells obtained increased adhesion properties and formed more conjugates with target cells. To increase the success of NK cell-based therapies of solid tumors, it is of great importance to promote their homing to the tumor site. In this study, we show that stable engineering of human primary NK cells to express a chemokine receptor thereby enhancing their migration is a promising strategy to improve anti-tumor responses following adoptive transfer of NK cells.

Licensed human natural killer cells aid dendritic cell maturation via TNFSF14/LIGHT

PubMed Central

Holmes, Tim D.; Wilson, Erica B.; Black, Emma V. I.; Benest, Andrew V.; Vaz, Candida; Tan, Betty; Tanavde, Vivek M.; Cook, Graham P.

2014-01-01

Interactions between natural killer (NK) cells and dendritic cells (DCs) aid DC maturation and promote T-cell responses. Here, we have analyzed the response of human NK cells to tumor cells, and we identify a pathway by which NK–DC interactions occur. Gene expression profiling of tumor-responsive NK cells identified the very rapid induction of TNF superfamily member 14 [TNFSF14; also known as homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT)], a cytokine implicated in the enhancement of antitumor responses. TNFSF14 protein expression was induced by three primary mechanisms of NK cell activation, namely, via the engagement of CD16, by the synergistic activity of multiple target cell-sensing NK-cell activation receptors, and by the cytokines IL-2 and IL-15. For antitumor responses, TNFSF14 was preferentially produced by the licensed NK-cell population, defined by the expression of inhibitory receptors specific for self-MHC class I molecules. In contrast, IL-2 and IL-15 treatment induced TNFSF14 production by both licensed and unlicensed NK cells, reflecting the ability of proinflammatory conditions to override the licensing mechanism. Importantly, both tumor- and cytokine-activated NK cells induced DC maturation in a TNFSF14-dependent manner. The coupling of TNFSF14 production to tumor-sensing NK-cell activation receptors links the tumor immune surveillance function of NK cells to DC maturation and adaptive immunity. Furthermore, regulation by NK cell licensing helps to safeguard against TNFSF14 production in response to healthy tissues. PMID:25512551

Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

PubMed Central

Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

2010-01-01

Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse.

PubMed

Huang, Yu; Chen, Zhiying; Jang, Joon Hee; Baig, Mirza S; Bertolet, Grant; Schroeder, Casey; Huang, Shengjian; Hu, Qian; Zhao, Yong; Lewis, Dorothy E; Qin, Lidong; Zhu, Michael Xi; Liu, Dongfang

2018-04-18

The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. We sought to determine the effect of PD-1 signaling on NK cells. PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

NK cells link obesity-induced adipose stress to inflammation and insulin resistance.

PubMed

Wensveen, Felix M; Jelenčić, Vedrana; Valentić, Sonja; Šestan, Marko; Wensveen, Tamara Turk; Theurich, Sebastian; Glasner, Ariella; Mendrila, Davor; Štimac, Davor; Wunderlich, F Thomas; Brüning, Jens C; Mandelboim, Ofer; Polić, Bojan

2015-04-01

An important cause of obesity-induced insulin resistance is chronic systemic inflammation originating in visceral adipose tissue (VAT). VAT inflammation is associated with the accumulation of proinflammatory macrophages in adipose tissue, but the immunological signals that trigger their accumulation remain unknown. We found that a phenotypically distinct population of tissue-resident natural killer (NK) cells represented a crucial link between obesity-induced adipose stress and VAT inflammation. Obesity drove the upregulation of ligands of the NK cell-activating receptor NCR1 on adipocytes; this stimulated NK cell proliferation and interferon-γ (IFN-γ) production, which in turn triggered the differentiation of proinflammatory macrophages and promoted insulin resistance. Deficiency of NK cells, NCR1 or IFN-γ prevented the accumulation of proinflammatory macrophages in VAT and greatly ameliorated insulin sensitivity. Thus NK cells are key regulators of macrophage polarization and insulin resistance in response to obesity-induced adipocyte stress.

Microwave-induced Apoptosis and Cytotoxicity of NK Cells through ERK1/2 Signaling.

PubMed

Zhao, Li; Li, Jing; Hao, Yan Hui; Gao, Ya Bing; Wang, Shui Ming; Zhang, Jing; Dong, Ji; Zhou, Hong Mei; Liu, Shu Chen; Peng, Rui Yun

2017-05-01

To investigate microwave-induced morphological and functional injury of natural killer (NK) cells and uncover their mechanisms. NK-92 cells were exposed to 10, 30, and 50 mW/cm2 microwaves for 5 min. Ultrastructural changes, cellular apoptosis and cell cycle regulation were detected at 1 h and 24 h after exposure. Cytotoxic activity was assayed at 1 h after exposure, while perforin and NKG2D expression were detected at 1 h, 6 h, and 12 h after exposure. To clarify the mechanisms, phosphorylated ERK (p-ERK) was detected at 1 h after exposure. Moreover, microwave-induced cellular apoptosis and cell cycle regulation were analyzed after blockade of ERK signaling by using U0126. Microwave-induced morphological and ultrastructural injury, dose-dependent apoptosis (P < 0.001) and cell cycle arrest (P < 0.001) were detected at 1 h after microwave exposure. Moreover, significant apoptosis was still detected at 24 h after 50 mW/cm2 microwave exposure (P < 0.01). In the 30 mW/cm2 microwave exposure model, microwaves impaired the cytotoxic activity of NK-92 cells at 1 h and down regulated perforin protein both at 1 h and 6 h after exposure (P < 0.05). Furthermore, p-ERK was down regulated at 1 h after exposure (P < 0.05), while ERK blockade significantly promoted microwave-induced apoptosis (P < 0.05) and downregulation of perforin (P < 0.01). Microwave dose-dependently induced morphological and functional injury in NK-92 cells, possibly through ERK-mediated regulation of apoptosis and perforin expression. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Expression of retinoid-related orphan receptor (ROR)γt on NK22 cells in the peripheral blood and uterine endometrium of women with unexplained recurrent pregnancy loss and unexplained infertility.

PubMed

Fuchinoue, Kohei; Fukui, Atsushi; Chiba, Hitomi; Kamoi, Mai; Funamizu, Ayano; Taima, Ayako; Fukuhara, Rie; Mizunuma, Hideki

2016-11-01

Recently, NK22 cells, a subset of interleukin (IL)-22-producing natural killer (NK) cells, were identified. We have previously reported the higher percentage of NK22 cells in women suffering recurrent pregnancy loss (RPL). Moreover, we have also reported lower expression of NKp46, a kind of natural cytotoxicity receptor (NCR), on NK cells and the changes of NK cell producing cytokines in women who experience RPL. NK22 cells express NCRs, such as NKp44 or NKp46. Retinoid-related orphan receptor γt (RORγt) is known as a regulator of NK22 cells; however, in NK22 cells of peripheral blood (PB) and the uterine endometrium (UE), the relationship between NCRs and RORγt is unclear. We investigate RORγt expression NK22 cells in the PB and UE of women with unexplained infertility (uI) or unexplained RPL (uRPL). Lymphocytes were extracted from PB and UE, derived from women with uI or uRPL. Expression of RORγt and NCRs in NK cells and NK cell-produced cytokines were analyzed by flow cytometry. CD56 + /NKp46 + /RORγt + cells were positively correlated with CD56 + /IL-22 + cells in both PB and UE. CD56 bright /NKp46 bright /RORγt + cells were significantly higher in uRPL than in uI, and endometrial CD56 bright /NKp46 bright /RORγt + cells were positively correlated with PB. In UE, CD56 bright /RORγt + cells were negatively correlated with CD56 bright /interferon-γ + and CD56 bright /tumor necrosis factor-α + cells of uRPL. RORγt may be associated with NK22 cells in reproduction. Particularly, higher expression of RORγt may be associated with elevated NK22 cells in uRPL. © 2016 Japan Society of Obstetrics and Gynecology.

Interactions between human mesenchymal stem cells and natural killer cells.

PubMed

Sotiropoulou, Panagiota A; Perez, Sonia A; Gritzapis, Angelos D; Baxevanis, Constantin N; Papamichail, Michael

2006-01-01

Mesenchymal stem cells (MSCs) are multipotent progenitor cells representing an attractive therapeutic tool for regenerative medicine. They possess unique immunomodulatory properties, being capable of suppressing T-cell responses and modifying dendritic cell differentiation, maturation, and function, whereas they are not inherently immunogenic, failing to induce alloreactivity to T cells and freshly isolated natural killer (NK) cells. To clarify the generation of host immune responses to implanted MSCs in tissue engineering and their potential use as immunosuppressive elements, the effect of MSCs on NK cells was investigated. We demonstrate that at low NK-to-MSC ratios, MSCs alter the phenotype of NK cells and suppress proliferation, cytokine secretion, and cyto-toxicity against HLA-class I- expressing targets. Some of these effects require cell-to-cell contact, whereas others are mediated by soluble factors, including transforming growth factor-beta1 and prostaglandin E2, suggesting the existence of diverse mechanisms for MSC-mediated NK-cell suppression. On the other hand, MSCs are susceptible to lysis by activated NK cells. Overall, these data improve our knowledge of interactions between MSCs and NK cells and consequently of their effect on innate immune responses and their contribution to the regulation of adaptive immunity, graft rejection, and cancer immunotherapy.

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.

A Systems Biology Approach Reveals that Tissue Tropism to West Nile Virus Is Regulated by Antiviral Genes and Innate Immune Cellular Processes

PubMed Central

Suthar, Mehul S.; Brassil, Margaret M.; Blahnik, Gabriele; McMillan, Aimee; Ramos, Hilario J.; Proll, Sean C.; Belisle, Sarah E.; Katze, Michael G.; Gale, Michael

2013-01-01

The actions of the RIG-I like receptor (RLR) and type I interferon (IFN) signaling pathways are essential for a protective innate immune response against the emerging flavivirus West Nile virus (WNV). In mice lacking RLR or IFN signaling pathways, WNV exhibits enhanced tissue tropism, indicating that specific host factors of innate immune defense restrict WNV infection and dissemination in peripheral tissues. However, the immune mechanisms by which the RLR and IFN pathways coordinate and function to impart restriction of WNV infection are not well defined. Using a systems biology approach, we defined the host innate immune response signature and actions that restrict WNV tissue tropism. Transcriptional profiling and pathway modeling to compare WNV-infected permissive (spleen) and nonpermissive (liver) tissues showed high enrichment for inflammatory responses, including pattern recognition receptors and IFN signaling pathways, that define restriction of WNV replication in the liver. Assessment of infected livers from Mavs−/−×Ifnar−/− mice revealed the loss of expression of several key components within the natural killer (NK) cell signaling pathway, including genes associated with NK cell activation, inflammatory cytokine production, and NK cell receptor signaling. In vivo analysis of hepatic immune cell infiltrates from WT mice demonstrated that WNV infection leads to an increase in NK cell numbers with enhanced proliferation, maturation, and effector action. In contrast, livers from Mavs−/−×Ifnar−/− infected mice displayed reduced immune cell infiltration, including a significant reduction in NK cell numbers. Analysis of cocultures of dendritic and NK cells revealed both cell-intrinsic and -extrinsic roles for the RLR and IFN signaling pathways to regulate NK cell effector activity. Taken together, these observations reveal a complex innate immune signaling network, regulated by the RLR and IFN signaling pathways, that drives tissue-specific antiviral effector gene expression and innate immune cellular processes that control tissue tropism to WNV infection. PMID:23544010

Tissue-Specific Education of Decidual NK Cells

PubMed Central

Xiong, Shiqiu; Kennedy, Philippa R.; Gardner, Lucy; Farrell, Lydia E.; Chazara, Olympe; Ivarsson, Martin A.; Hiby, Susan E.; Colucci, Francesco; Moffett, Ashley

2015-01-01

During human pregnancy, fetal trophoblast cells invade the decidua and remodel maternal spiral arteries to establish adequate nutrition during gestation. Tissue NK cells in the decidua (dNK) express inhibitory NK receptors (iNKR) that recognize allogeneic HLA-C molecules on trophoblast. Where this results in excessive dNK inhibition, the risk of pre-eclampsia or growth restriction is increased. However, the role of maternal, self–HLA-C in regulating dNK responsiveness is unknown. We investigated how the expression and function of five iNKR in dNK is influenced by maternal HLA-C. In dNK isolated from women who have HLA-C alleles that carry a C2 epitope, there is decreased expression frequency of the cognate receptor, KIR2DL1. In contrast, women with HLA-C alleles bearing a C1 epitope have increased frequency of the corresponding receptor, KIR2DL3. Maternal HLA-C had no significant effect on KIR2DL1 or KIR2DL3 in peripheral blood NK cells (pbNK). This resulted in a very different KIR repertoire for dNK capable of binding C1 or C2 epitopes compared with pbNK. We also show that, although maternal KIR2DL1 binding to C2 epitope educates dNK cells to acquire functional competence, the effects of other iNKR on dNK responsiveness are quite different from those in pbNK. This provides a basis for understanding how dNK responses to allogeneic trophoblast affect the outcome of pregnancy. Our findings suggest that the mechanisms that determine the repertoire of iNKR and the effect of self-MHC on NK education may differ in tissue NK cells compared with pbNK. PMID:26320253

Genetic deletion of Cxcl14 in mice alters uterine NK cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Qichen; Graduate School of the Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan, Beijing 100049; Chen, Hua

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 populationmore » 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.« less

The DNA methylation profile of activated human natural killer cells.

PubMed

Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

2016-05-03

Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states.

The role of Gαs in activation of NK92-MI cells by neuropeptide substance P.

PubMed

Diandong, Hou; Kefeng, Sun; Weixin, Fu; Moran, Wang; Jiahui, Wang; Zaifu, Liang

2014-02-01

Substance P (SP) is well known for its immunoregulatory influence on NK cells. The biological actions of SP are mediated primarily through the high-affinity neurokinin-1 receptor (NK-1R), a G protein-coupled receptor (GPCR). Receptor binding triggers a cAMP signaling pathway and intracellular levels of cAMP are regulated via Gαs and Gαi. In this study NF449, a Gαs-selective G protein antagonist, was used to study the role of Gαs in the activation of NK92-MI cells by SP. Results show that 10(-12)M SP enhances the expression of Gαs and Gαi3 in NK92-MI cells promoting a cytotoxic phenotype characterized by expression of perforin and granzyme B. Development of a cytotoxic phenotype in NK92-MI cells stimulated with SP is blunted by inhibition of Gαs by NF449. In summary, SP signaling through NK-1R promotes a cytotoxic phenotype in NK92-MI cells characterized by upregulation of both Gαs and Gαi3. NF449 inhibits Gαs, blunts SP-induced expression of perforin and granzyme B, and represents a potential therapeutic avenue for reducing NK-cell mediated cytotoxicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Clinical Grade Purification and Expansion of NK Cell Products for an Optimized Manufacturing Protocol

PubMed Central

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 × 108 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. PMID:23730623

Distribution pattern of tachykinin NK2 receptors in human colon: involvement in the regulation of intestinal motility.

PubMed

Jaafari, Nadia; Khomitch-Baud, Alexandra; Christen, Marie-Odile; Julé, Yvon

2007-07-20

Although a number of pharmacological studies have shown the involvement of tachykinin type 2 receptors (NK2r) in the regulation of human colonic motility, few data are available so far on their pattern of expression. In this study this pattern was investigated in the myenteric plexuses, the longitudinal and circular muscle layers (external muscular layers), and the interstitial cells of Cajal (ICCs) using confocal microscopy immunofluorescence methods. NK2r immunoreactivity (NK2r-IR) was detected in the soma of myenteric neurons and in nerve varicosities located in myenteric plexuses as well as in external muscular layers. Colocalization analysis of NK2r-IR and synaptophysin-IR, showed significant regional differences in the distribution of NK2r-expressing nerve varicosities, the rate of occurrence was found to be 56.08% +/- 3% (mean +/- SE) in the external muscular layers and 30.22% +/- 1% (mean +/- SE) in the myenteric plexuses. NK2r-IR was found in membranes of most muscle cells previously incubated with a selective NK2r agonist, [beta-Ala(8)] neurokinin A fragment 4-10, at 4 degrees C, and then mainly relocated in the cytoplasm when heated to 37 degrees C. A number of NK2r-IR nerve varicosities were close to NK2r-expressing neurons and muscle cells. Some of NK2r-expressing neurons and nerves were tachykinin-IR. No NK2r-IR was detected in ICCs. The present data indicate that presynaptic and postsynaptic neuroneuronal and neuromuscular regulatory processes mediated by tachykinins via NK2r may occur for modulating human colonic motility.

Role of Natural Killer Cells in the Innate Immune System After Intraportal Islet Transplantation in Mice.

PubMed

Saeki, Y; Ishiyama, K; Ishida, N; Tanaka, Y; Ohdan, H

Both liver natural killer (NK) and NK T cells of the innate immune system play a crucial role in islet graft loss after intraportal islet transplantation, although a relationship between NK and NK T cells in islet loss has not been proven. In this study, we investigated the role of NK cells in the innate immune system in islet graft loss after intraportal islet transplantation. To investigate the involvement of liver NK cells in islet destruction, we assessed the differences in graft survival after intraportal islet transplantation between CD1d -/- diabetic mice and NK cell-depleted CD1d -/- diabetic mice. The transplantation of 400 islets into the liver was sufficient to reverse hyperglycemia in wild-type diabetic mice (100%, 4/4). However, normoglycemia could not be achieved when 200 islets were transplanted (0%, 0/4). In contrast, intraportal transplantation of 200 islets in NK cell-depleted CD1d -/- diabetic mice ameliorated hyperglycemia in 71% of cases (5/7), whereas transplantation of the same number of islets in CD1d -/- diabetic mice did not (0%, 0/4). Histologic findings also confirmed that intact islets were observed in NK cell-depleted CD1d -/- diabetic mice, but were difficult to observe in CD1d -/- diabetic mice. The involvement of liver NK cells in the innate immune system related to islet graft loss after intraportal islet transplantation is revealed by improved graft survival and function in NK cell-depleted CD1d -/- diabetic mice. Our data reveal that regulation of NK cell activity is particularly important when insufficient islet numbers are used for transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

Elevated Levels of Serum Vascular Endothelial Growth Factor-A Are Not Related to NK Cell Parameters in Recurrent IVF Failure.

PubMed

Bansal, Rhea; Ford, Brian; Bhaskaran, Shree; Thum, Meenyau; Bansal, Amolak

2017-01-01

Vascular Endothelial Growth Factor and NK cells have an interrelated role in angiogenesis that is critical for placentation and success of in vitro fertilization. An attempt was made to assess a possible relationship between the two in this study. A case control study was performed comparing the serum levels of VEGF-A and its receptor VEGF-R1 with levels of NK cells, activated NK cells and NK cytotoxicity in 62 women with Repeated Implantation Failure (RIF). The healthy control group consisted of 72 women of similar age, without known issues in achieving pregnancy or evidence of autoimmunity. Levels of VEGF-A and VEGF-R1 were quantified by ELISA methods with standard curve interpolation. NK cell subsets were determined with flow cytometry using fluorescent-tagged anti-CD56, anti-CD16, anti-CD3 and anti-CD69. NK cytotoxicity was performed by incubating peripheral blood mononuclear cells and K562 cultured cells with propidium iodide, steroid, intralipid and intravenous immunoglobulin, using previously described methods. Statistical analysis involved Mann-Whitney-U and Spearman's rank correlation testing with p-values defined as <0.05. It was found that VEGF-A levels were significantly raised in women with RIF compared to healthy controls (362.9 vs . 171.6 pg/ml , p<0.0001), with no difference in VEGF-R1 levels between groups (1499 vs . 1202 pg/ml , p=0.4082). There was no correlation between VEGF-A or VEGF-R1 and the absolute levels of circulating NK cells, CD69 activated NK cells or NK cytotoxicity. The absence of correlation between VEGF-A or VEGF-R1 and NK cells suggests VEGF secretion and regulation is independent of NK cell activity in RIF.

Dysregulated cellular functions and cell stress pathways provide critical cues for activating and targeting natural killer cells to transformed and infected cells.

PubMed

Raulet, David H; Marcus, Assaf; Coscoy, Laurent

2017-11-01

Natural killer (NK) cells recognize and kill cancer cells and infected cells by engaging cell surface ligands that are induced preferentially or exclusively on these cells. These ligands are recognized by activating receptors on NK cells, such as NKG2D. In addition to activation by cell surface ligands, the acquisition of optimal effector activity by NK cells is driven in vivo by cytokines and other signals. This review addresses a developing theme in NK cell biology: that NK-activating ligands on cells, and the provision of cytokines and other signals that drive high effector function in NK cells, are driven by abnormalities that arise from transformation or the infected state. The pathways include genomic damage, which causes self DNA to be exposed in the cytosol of affected cells, where it activates the DNA sensor cGAS. The resulting signaling induces NKG2D ligands and also mobilizes NK cell activation. Other key pathways that regulate NKG2D ligands include PI-3 kinase activation, histone acetylation, and the integrated stress response. This review summarizes the roles of these pathways and their relevance in both viral infections and cancer. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Interleukin (IL)-18 Binding Protein Deficiency Disrupts Natural Killer Cell Maturation and Diminishes Circulating IL-18

PubMed Central

Harms, Robert Z.; Creer, Austin J.; Lorenzo-Arteaga, Kristina M.; Ostlund, Katie R.; Sarvetnick, Nora E.

2017-01-01

The cytokine interleukin (IL)-18 is a crucial amplifier of natural killer (NK) cell function. IL-18 signaling is regulated by the inhibitory effects of IL-18 binding protein (IL-18BP). Using mice deficient in IL-18BP (IL-18BPKO), we investigated the impact of mismanaged IL-18 signaling on NK cells. We found an overall reduced abundance of splenic NK cells in the absence of IL-18BP. Closer examination of NK cell subsets in spleen and bone marrow using CD27 and CD11b expression revealed that immature NK cells were increased in abundance, while the mature population of NK cells was reduced. Also, NK cells were polarized to greater production of TNF-α, while dedicated IFN-γ producers were reduced. A novel subset of IL-18 receptor α− NK cells contributed to the expansion of immature NK cells in IL-18BPKO mice. Splenocytes cultured with IL-18 resulted in alterations similar to those observed in IL-18BP deficiency. NK cell changes were associated with significantly reduced levels of circulating plasma IL-18. However, IL-18BPKO mice exhibited normal weight gain and responded to LPS challenge with a >10-fold increase in IFN-γ compared to wild type. Finally, we identified that the source of splenic IL-18BP was among dendritic cells/macrophage localized to the T cell-rich regions of the spleen. Our results demonstrate that IL-18BP is required for normal NK cell abundance and function and also contributes to maintaining steady-state levels of circulating IL-18. Thus, IL-18BP appears to have functions suggestive of a carrier protein, not just an inhibitor. PMID:28900426

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

PubMed

Song, Yanfang; Gan, Yu; Wang, Qing; Meng, Zihong; Li, Guohua; Shen, Yuling; Wu, Yufeng; Li, Peiying; Yao, Ming; Gu, Jianren; Tu, Hong

2017-04-01

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

Modification of P-selectin glycoprotein ligand-1 with a natural killer cell-restricted sulfated lactosamine creates an alternate ligand for L-selectin

PubMed Central

André, Pascale; Spertini, Olivier; Guia, Sophie; Rihet, Pascal; Dignat-George, Françoise; Brailly, Hervé; Sampol, José; Anderson, Paul J.; Vivier, Eric

2000-01-01

Natural killer (NK) cells are components of the innate immune system that can recognize and kill virally infected cells, tumor cells, and allogeneic cells without prior sensitization. NK cells also elaborate cytokines (e.g., interferon-γ and tumor necrosis factor-α) and chemokines (e.g., macrophage inflammatory protein-1α) that promote the acquisition of antigen-specific immunity. NK cell differentiation is accompanied by the cell surface expression of a mucin-like glycoprotein bearing an NK cell-restricted keratan sulfate-related lactosamine carbohydrate, the PEN5 epitope. Here, we report that PEN5 is a post-translational modification of P-selectin glycoprotein ligand-1 (PSGL-1). The PEN5 epitope creates on PSGL-1 a unique binding site for L-selectin, which is independent of PSGL-1 tyrosine sulfation. On the surface of NK cells, the expression of PEN5 is coordinated with the disappearance of L-selectin and the up-regulation of Killer cell Ig-like Receptors (KIR). These results indicate that NK cell differentiation is accompanied by the acquisition of a unique carbohydrate, PEN5, that can serve as part of a combination code to deliver KIR+ NK cells to specific tissues. PMID:10725346

Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction.

PubMed

Mathew, Stephen O; Chaudhary, Pankaj; Powers, Sheila B; Vishwanatha, Jamboor K; Mathew, Porunelloor A

2016-10-18

Prostate cancer is the most common type of cancer diagnosed and the second leading cause of cancer-related death in American men. Natural Killer (NK) cells are the first line of defense against cancer and infections. NK cell function is regulated by a delicate balance between signals received through activating and inhibitory receptors. Previously, we identified Lectin-like transcript-1 (LLT1/OCIL/CLEC2D) as a counter-receptor for the NK cell inhibitory receptor NKRP1A (CD161). Interaction of LLT1 expressed on target cells with NKRP1A inhibits NK cell activation. In this study, we have found that LLT1 was overexpressed on prostate cancer cell lines (DU145, LNCaP, 22Rv1 and PC3) and in primary prostate cancer tissues both at the mRNA and protein level. We further showed that LLT1 is retained intracellularly in normal prostate cells with minimal cell surface expression. Blocking LLT1 interaction with NKRP1A by anti-LLT1 mAb on prostate cancer cells increased the NK-mediated cytotoxicity of prostate cancer cells. The results indicate that prostate cancer cells may evade immune attack by NK cells by expressing LLT1 to inhibit NK cell-mediated cytolytic activity through LLT1-NKRP1A interaction. Blocking LLT1-NKRP1A interaction will make prostate cancer cells susceptible to killing by NK cells and therefore may be a new therapeutic option for treatment of prostate cancer.

Oxygen Modulates Human Decidual Natural Killer Cell Surface Receptor Expression and Interactions with Trophoblasts1

PubMed Central

Wallace, Alison E.; Goulwara, Sonu S.; Whitley, Guy S.; Cartwright, Judith E.

2014-01-01

Decidual natural killer (dNK) cells have been shown to both promote and inhibit trophoblast behavior important for decidual remodeling in pregnancy and have a distinct phenotype compared to peripheral blood NK cells. We investigated whether different levels of oxygen tension, mimicking the physiological conditions of the decidua in early pregnancy, altered cell surface receptor expression and activity of dNK cells and their interactions with trophoblast. dNK cells were isolated from terminated first-trimester pregnancies and cultured in oxygen tensions of 3%, 10%, and 21% for 24 h. Cell surface receptor expression was examined by flow cytometry, and the effects of secreted factors in conditioned medium (CM) on the trophoblast cell line SGHPL-4 were assessed in vitro. SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 10% were significantly more invasive (P < 0.05) and formed endothelial-like networks to a greater extent (P < 0.05) than SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 3% or 21%. After 24 h, a lower percentage of dNK cells expressed CD56 at 21% oxygen (P < 0.05), and an increased percentage of dNK cells expressed NKG2D at 10% oxygen (P < 0.05) compared to other oxygen tensions, with large patient variation. This study demonstrates dNK cell phenotype and secreted factors are modulated by oxygen tension, which induces changes in trophoblast invasion and endovascular-like differentiation. Alterations in dNK cell surface receptor expression and secreted factors at different oxygen tensions may represent regulation of function within the decidua during the first trimester of pregnancy. PMID:25232021

Interleukin (IL)-23 Stimulates IFN-γ Secretion by CD56bright Natural Killer Cells and Enhances IL-18-Driven Dendritic Cells Activation.

PubMed

Ziblat, Andrea; Nuñez, Sol Y; Raffo Iraolagoitia, Ximena Lucía; Spallanzani, Raúl German; Torres, Nicolás I; Sierra, Jessica M; Secchiari, Florencia; Domaica, Carolina I; Fuertes, Mercedes B; Zwirner, Norberto W

2017-01-01

Interleukin (IL)-23 is a member of the IL-12 family of cytokines that, as the other members of this family, is secreted by monocytes, macrophages, and dendritic cells (DC) upon recognition of bacterial, viral, and fungal components. IL-23 is critical during immunity against acute infections, and it is also involved in the development of autoimmune diseases. Although immunoregulatory effects of IL-23 on mouse natural killer (NK) cells have been described, the effect of IL-23 on human NK cells remains ill-defined. In this study, we observed that monocytes stimulated with LPS secreted IL-23 and that blockade of this cytokine during monocyte and NK cell coculture led to a diminished production of IFN-γ by NK cells. Accordingly, rIL-23-induced NK cell activation and stimulated IFN-γ production by CD56 bright NK cells. This effect involved MEK1/MEK2, JNK, PI3K, mammalian target of rapamycin, and NF-κB, but not STAT-1, STAT-3, nor p38 MAPK pathways. Moreover, while NK cell-mediated cytotoxicity remained unaltered, antibody-dependent cellular cytotoxicity (ADCC) was enhanced after IL-23 stimulation. In addition, IL-23 displayed a synergistic effect with IL-18 for IFN-γ production by both CD56 bright and CD56 dim NK cells, and this effect was due to a priming effect of IL-23 for IL-18 responsiveness. Furthermore, NK cells pre-stimulated with IL-18 promoted an increase in CD86 expression and IL-12 secretion by DC treated with LPS, and IL-23 potentiated these effects. Moreover, IL-23-driven enhancement of NK cell "helper" function was dependent on NK cell-derived IFN-γ. Therefore, our results suggest that IL-23 may trigger NK cell-mediated "helper" effects on adaptive immunity, shaping T cell responses during different pathological situations through the regulation of DC maturation.

Interleukin (IL)-23 Stimulates IFN-γ Secretion by CD56bright Natural Killer Cells and Enhances IL-18-Driven Dendritic Cells Activation

PubMed Central

Ziblat, Andrea; Nuñez, Sol Y.; Raffo Iraolagoitia, Ximena Lucía; Spallanzani, Raúl German; Torres, Nicolás I.; Sierra, Jessica M.; Secchiari, Florencia; Domaica, Carolina I.; Fuertes, Mercedes B.; Zwirner, Norberto W.

2018-01-01

Interleukin (IL)-23 is a member of the IL-12 family of cytokines that, as the other members of this family, is secreted by monocytes, macrophages, and dendritic cells (DC) upon recognition of bacterial, viral, and fungal components. IL-23 is critical during immunity against acute infections, and it is also involved in the development of autoimmune diseases. Although immunoregulatory effects of IL-23 on mouse natural killer (NK) cells have been described, the effect of IL-23 on human NK cells remains ill-defined. In this study, we observed that monocytes stimulated with LPS secreted IL-23 and that blockade of this cytokine during monocyte and NK cell coculture led to a diminished production of IFN-γ by NK cells. Accordingly, rIL-23-induced NK cell activation and stimulated IFN-γ production by CD56bright NK cells. This effect involved MEK1/MEK2, JNK, PI3K, mammalian target of rapamycin, and NF-κB, but not STAT-1, STAT-3, nor p38 MAPK pathways. Moreover, while NK cell-mediated cytotoxicity remained unaltered, antibody-dependent cellular cytotoxicity (ADCC) was enhanced after IL-23 stimulation. In addition, IL-23 displayed a synergistic effect with IL-18 for IFN-γ production by both CD56bright and CD56dim NK cells, and this effect was due to a priming effect of IL-23 for IL-18 responsiveness. Furthermore, NK cells pre-stimulated with IL-18 promoted an increase in CD86 expression and IL-12 secretion by DC treated with LPS, and IL-23 potentiated these effects. Moreover, IL-23-driven enhancement of NK cell “helper” function was dependent on NK cell-derived IFN-γ. Therefore, our results suggest that IL-23 may trigger NK cell-mediated “helper” effects on adaptive immunity, shaping T cell responses during different pathological situations through the regulation of DC maturation. PMID:29403472

TLR expression and NK cell activation after human yellow fever vaccination.

PubMed

Neves, Patrícia Cristina da Costa; Matos, Denise Cristina de Souza; Marcovistz, Rugimar; Galler, Ricardo

2009-09-18

The yellow fever vaccine is very effective with a single injection conferring protection for at least 10 years. Recent evidence suggests that the innate immune cells activated through Toll-like receptors (TLRs), are critical determinants of the robustness of the adaptive response. Therefore, we investigated the NK cell status in eight healthy volunteers after vaccination with YF 17DD virus. Shortly after vaccination, we observed increased expression of TLR-3 and TLR-9 in NK cells and markers such as CD69, HLA-DP-DQ-DR, CD38 and CD16. The up-regulation of CD69 was positively correlated with the presence of TLRs throughout the post-vaccination period and the circulating IFN-gamma was significantly augmented. These results suggest that TLRs may play an important role in NK cell activation during the immune response to vaccination, indicating a potential role for NK cells in helping the development of long-lasting protective memory.

Critical role of dendritic cell-derived IL-27 in antitumor immunity through regulating the recruitment and activation of NK and NKT cells.

PubMed

Wei, Jun; Xia, Siyuan; Sun, Huayan; Zhang, Song; Wang, Jingya; Zhao, Huiyuan; Wu, Xiaoli; Chen, Xi; Hao, Jianlei; Zhou, Xinglong; Zhu, Zhengmao; Gao, Xiang; Gao, Jian-xin; Wang, Puyue; Wu, Zhenzhou; Zhao, Liqing; Yin, Zhinan

2013-07-01

Critical roles of IL-27 in autoimmune diseases and infections have been reported; however, the contribution of endogenous IL-27 to tumor progression remains elusive. In this study, by using IL-27p28 conditional knockout mice, we demonstrate that IL-27 is critical in protective immune response against methyl-cholanthrene-induced fibrosarcoma and transplanted B16 melanoma, and dendritic cells (DCs) are the primary source. DC-derived IL-27 is required for shaping tumor microenvironment by inducing CXCL-10 expression in myeloid-derived suppressor cells and regulating IL-12 production from DCs, which lead to the recruitment and activation of NK and NKT cells resulting in immunological control of tumors. Indeed, reconstitution of IL-27 or CXCL-10 in tumor site significantly inhibits tumor growth and restores the number and activation of NK and NKT cells. In summary, our study identifies a previous unknown critical role of DC-derived IL-27 in NK and NKT cell-dependent antitumor immunity through shaping tumor microenvironment, and sheds light on developing novel therapeutic approaches based on IL-27.

Uterine NK Cells Are Critical in Shaping DC Immunogenic Functions Compatible with Pregnancy Progression

PubMed Central

Freitag, Nancy; Otto, Teresa; Thijssen, Victor L. J. L.; Moschansky, Petra; von Kwiatkowski, Petra; Klapp, Burghard F.; Winterhager, Elke; Bauersachs, Stefan; Blois, Sandra M.

2012-01-01

Dendritic cell (DC) and natural killer (NK) cell interactions are important for the regulation of innate and adaptive immunity, but their relevance during early pregnancy remains elusive. Using two different strategies to manipulate the frequency of NK cells and DC during gestation, we investigated their relative impact on the decidualization process and on angiogenic responses that characterize murine implantation. Manipulation of the frequency of NK cells, DC or both lead to a defective decidual response characterized by decreased proliferation and differentiation of stromal cells. Whereas no detrimental effects were evident upon expansion of DC, NK cell ablation in such expanded DC mice severely compromised decidual development and led to early pregnancy loss. Pregnancy failure in these mice was associated with an unbalanced production of anti-angiogenic signals and most notably, with increased expression of genes related to inflammation and immunogenic activation of DC. Thus, NK cells appear to play an important role counteracting potential anomalies raised by DC expansion and overactivity in the decidua, becoming critical for normal pregnancy progression. PMID:23056436

Uterine NK cells are critical in shaping DC immunogenic functions compatible with pregnancy progression.

PubMed

Tirado-González, Irene; González, Irene Tirado; Barrientos, Gabriela; Freitag, Nancy; Otto, Teresa; Thijssen, Victor L J L; Moschansky, Petra; von Kwiatkowski, Petra; Klapp, Burghard F; Winterhager, Elke; Bauersachs, Stefan; Blois, Sandra M

2012-01-01

Dendritic cell (DC) and natural killer (NK) cell interactions are important for the regulation of innate and adaptive immunity, but their relevance during early pregnancy remains elusive. Using two different strategies to manipulate the frequency of NK cells and DC during gestation, we investigated their relative impact on the decidualization process and on angiogenic responses that characterize murine implantation. Manipulation of the frequency of NK cells, DC or both lead to a defective decidual response characterized by decreased proliferation and differentiation of stromal cells. Whereas no detrimental effects were evident upon expansion of DC, NK cell ablation in such expanded DC mice severely compromised decidual development and led to early pregnancy loss. Pregnancy failure in these mice was associated with an unbalanced production of anti-angiogenic signals and most notably, with increased expression of genes related to inflammation and immunogenic activation of DC. Thus, NK cells appear to play an important role counteracting potential anomalies raised by DC expansion and overactivity in the decidua, becoming critical for normal pregnancy progression.

Neurokinin-1 receptor agonists bias therapeutic dendritic cells to induce type 1 immunity by licensing host dendritic cells to produce IL-12

PubMed Central

Janelsins, Brian M.; Sumpter, Tina L.; Tkacheva, Olga A.; Rojas-Canales, Darling M.; Erdos, Geza; Mathers, Alicia R.; Shufesky, William J.; Storkus, Walter J.; Falo, Louis D.; Morelli, Adrian E.; Larregina, Adriana T.

2013-01-01

Substance-P and hemokinin-1 are proinflammatory neuropeptides with potential to promote type 1 immunity through agonistic binding to neurokinin-1 receptor (NK1R). Dendritic cells (DCs) are professional antigen-presenting cells that initiate and regulate the outcome of innate and adaptive immune responses. Immunostimulatory DCs are highly desired for the development of positive immunization techniques. DCs express functional NK1R; however, regardless of their potential DC-stimulatory function, the ability of NK1R agonists to promote immunostimulatory DCs remains unexplored. Here, we demonstrate that NK1R signaling activates therapeutic DCs capable of biasing type 1 immunity by inhibition of interleukin-10 (IL-10) synthesis and secretion, without affecting their low levels of IL-12 production. The potent type 1 effector immune response observed following cutaneous administration of NK1R-signaled DCs required their homing in skin-draining lymph nodes (sDLNs) where they induced inflammation and licensed endogenous-conventional sDLN-resident and -recruited inflammatory DCs to secrete IL-12. Our data demonstrate that NK1R signaling promotes immunostimulatory DCs, and provide relevant insight into the mechanisms used by neuromediators to regulate innate and adaptive immune responses. PMID:23365459

NK cells contribute to persistent airway inflammation and AHR during the later stage of RSV infection in mice.

PubMed

Long, Xiaoru; Xie, Jun; Zhao, Keting; Li, Wei; Tang, Wei; Chen, Sisi; Zang, Na; Ren, Luo; Deng, Yu; Xie, Xiaohong; Wang, Lijia; Fu, Zhou; Liu, Enmei

2016-10-01

RSV can lead to persistent airway inflammation and AHR and is intimately associated with childhood recurrent wheezing and asthma, but the underlying mechanisms remain unclear. There are high numbers of NK cells in the lung, which not only play important roles in the acute stage of RSV infection, but also are pivotal in regulating the pathogenesis of asthma. Therefore, in this study, we assumed that NK cells might contribute to persistent airway disease during the later stage of RSV infection. Mice were killed at serial time points after RSV infection to collect samples. Leukocytes in bronchoalveolar lavage fluid (BALF) were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. Cytokines were detected by ELISA, and NK cells were determined by flow cytometry. Rabbit anti-mouse asialo-GM-1 antibodies and resveratrol were used to deplete or suppress NK cells. Inflammatory cells in BALF, lung tissue damage and AHR were persistent for 60 days post-RSV infection. Type 2 cytokines and NK cells were significantly increased during the later stage of infection. When NK cells were decreased by the antibodies or resveratrol, type 2 cytokines, the persistent airway inflammation and AHR were all markedly reduced. NK cells can contribute to the RSV-associated persistent airway inflammation and AHR at least partially by promoting type 2 cytokines. Therefore, therapeutic targeting of NK cells may provide a novel approach to alleviating the recurrent wheezing subsequent to RSV infection.

Activation of natural killer cells by hepatitis C virus particles in vitro

PubMed Central

Farag, M M S; Weigand, K; Encke, J; Momburg, F

2011-01-01

Little is known about the ability of hepatitis C virus (HCV) to alter early innate immune responses in infected patients. Previous studies have shown that natural killer (NK) cells are functionally impaired after interaction of recombinant HCV glycoprotein E2 with the co-stimulatory CD81 molecule in vitro; however, the functional consequences of a prolonged contact of NK cells with HCV particles have remained unclear. We have examined the phenotypes of purified, interleukin-2-activated NK cells from healthy donors and HCV genotype 1b patients after culture for 5 days with HCV pseudoparticles (HCVpp) and serum samples containing HCV genotype 1b. NK cells from healthy donors and chronic HCV patients were found to up-regulate receptors associated with activation (NKp46, NKp44, NKp30, NKG2D), while NK receptors from the killer cell immunoglobulin-like receptor family (KIR/CD158), predominantly having an inhibitory function, were significantly down-modulated after culture in the presence of HCV particles compared with control cultures of NK cells. HCV-infected sera and HCVpp elicited significantly higher secretion of the NK effector lymphokines interferon-γ and tumour necrosis factor-α. Furthermore, HCV stimulated the cytotoxic potential of NK cells from normal donors and patients. The enhanced activation of NK cells after prolonged culture with HCVpp or HCV-containing sera for 5 days suggests that these innate effector cells may play an important role in viral control during early phases of HCV infection. PMID:21682720

The impact of ageing on natural killer cell function and potential consequences for health in older adults.

PubMed

Hazeldine, Jon; Lord, Janet M

2013-09-01

Forming the first line of defence against virally infected and malignant cells, natural killer (NK) cells are critical effector cells of the innate immune system. With age, significant impairments have been reported in the two main mechanisms by which NK cells confer host protection: direct cytotoxicity and the secretion of immunoregulatory cytokines and chemokines. In elderly subjects, decreased NK cell activity has been shown to be associated with an increased incidence and severity of viral infection, highlighting the clinical implications that age-associated changes in NK cell biology have on the health of older adults. However, is an increased susceptibility to viral infection the only consequence of these age-related changes in NK cell function? Recently, evidence has emerged that has shown that in addition to eliminating transformed cells, NK cells are involved in many other biological processes such as immune regulation, anti-microbial immune responses and the recognition and elimination of senescent cells, novel functions that involve NK-mediated cytotoxicity and/or cytokine production. Thus, the decrease in NK cell function that accompanies physiological ageing is likely to have wider implications for the health of older adults than originally thought. Here, we give a detailed description of the changes in NK cell biology that accompany human ageing and propose that certain features of the ageing process such as: (i) the increased reactivation rates of latent Mycobacterium tuberculosis, (ii) the slower resolution of inflammatory responses and (iii) the increased incidence of bacterial and fungal infection are attributable in part to an age-associated decline in NK cell function. Copyright © 2013 Elsevier B.V. All rights reserved.

Histaminergic regulation of NK-cells: protection against monocyte-induced apoptosis.

PubMed

Hansson, M; Asea, A; Hermodsson, S; Hellstrand, K

1996-08-01

Human natural killer (NK) cells (with CD3-/56+ phenotype) acquired features characteristic of apoptosis after incubation with autologous monocytes, as revealed by apoptotic nuclear morphology and degradation of DNA into oligonucleosomal fragments. The monocyte-induced apoptosis in NK-cells was prevented by the biogenic amine histamine at concentrations exceeding 0.1 microM. The protective effect of histamine was blocked by the H2-receptor (H2R) antagonist ranitidine but not by AH202399 A, a chemical control to ranitidine devoid of H2R affinity. It is concluded that histaminergic mechanisms may serve to protect NK cells from damage inflicted by products of the oxidative metabolism of monocytes.

Role of protein kinase C in TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells.

PubMed

Abraha, Abraham B; Rana, Krupa; Whalen, Margaret M

2010-11-01

Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposure of NK cells to tributyltin (TBT) greatly diminishes their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C(PKC) as well as MAPK activity. TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposure. TBT caused a 2–3-fold activation of PKC at concentrations ranging from 50 to 300 nM (16–98 ng/ml),indicating that activation of PKC occurs in response to TBT exposure. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells, validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that, in NK cells where PKC activation was blocked, there was no activation of the MAPK, p44/42 in response to TBT.However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including activation of p44/42 by TBT in NK cells.

Role of protein kinase C in the TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells

PubMed Central

Abraha, Abraham B.; Rana, Krupa; Whalen, Margaret M.

2010-01-01

Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposures of NK cells to tributyltin (TBT) greatly diminish their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in the NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C (PKC) as well as MAPK activity. The TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in the inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposures. TBT caused a 2–3 fold activation of PKC at concentrations ranging from 50–300 nM (16–98 ng/mL), indicating that activation of PKC occurs in response to TBT exposures. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that in NK cells where PKC activation was blocked there was no activation of the MAPK, p44/42 in response to TBT. However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including the activation of p44/42 by TBT in NK cells. PMID:20390410

Polymorphonuclear neutrophils and granulocytic myeloid-derived suppressor cells inhibit natural killer cell activity toward Aspergillus fumigatus.

PubMed

Mueller-Leisse, Johanna; Brueggemann, Sabrina; Bouzani, Maria; Schmitt, Anna-Lena; Einsele, Hermann; Loeffler, Juergen

2015-08-01

Invasive aspergillosis is a devastating infectious disease in immunocompromised patients. Besides neutrophils and macrophages, natural killer (NK) cells have recently emerged as important players in immunity to this infection. It was shown that NK cells comprise an essential role in the clearance of Aspergillus fumigatus (A. fumigatus) in neutropenic but not in nonneutropenic mice. However, the antifungal activity of NK cells and their regulation have not been fully characterized. In this study, we investigated the interplay between polymorphonuclear neutrophils (PMNs) or granulocyte myeloid-derived suppressor cells (Gr-MDSCs) with NK cells. Both cell types exhibited an equal inhibitory effect on NK cell activation through downregulation of NKp30 expression on the cell surface and cytotoxicity towards the cell line K562. Furthermore, we showed that NK cell activation and antifungal cytotoxicity were impaired when NK cells had been cultured in the presence of PMNs or Gr-MDSCs before fungal stimulation. Besides the reduced cytotoxicity a decreased release of interferon gamma (IFNγ), a key player in the clearance of an A. fumigatus infection, was observed. Thus, inhibition of NK cell activity by PMNs or Gr-MDSCs might impair an effective anti-fungal immune response during recovery from conditions such as hematopoietic stem cell transplantation. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Soluble HLA-G dampens CD94/NKG2A expression and function and differentially modulates chemotaxis and cytokine and chemokine secretion in CD56bright and CD56dim NK cells.

PubMed

Morandi, Fabio; Ferretti, Elisa; Castriconi, Roberta; Dondero, Alessandra; Petretto, Andrea; Bottino, Cristina; Pistoia, Vito

2011-11-24

Soluble HLA-G (sHLA-G) inhibits natural killer (NK) cell functions. Here, we investigated sHLA-G-mediated modulation of (1) chemokine receptor and NK receptor expression and function and (2) cytokine and chemokine secretion in CD56bright and CD56dim NK cells. sHLA-G-treated or untreated peripheral blood (PB) and tonsil NK cells were analyzed for chemokine receptor and NK receptor expression by flow cytometry. sHLA-G down-modulated (1) CXCR3 on PB and tonsil CD56bright and CD56dim, (2) CCR2 on PB and tonsil CD56bright, (3) CX3CR1 on PB CD56dim, (4) CXCR5 on tonsil CD56dim, and (5) CD94/NKG2A on PB and tonsil CD56brigh) and CD56dim NK cells. Such sHLA-G-mediated down-modulations were reverted by adding anti-HLA-G or anti-ILT2 mAbs. sHLA-G inhibited chemotaxis of (1) PB NK cells toward CXCL10, CXCL11, and CX3CL1 and (2) PB CD56bright NK cells toward CCL2 and CXCL10. IFN-γ secretion induced by NKp46 engagement was inhibited by NKG2A engagement in untreated but not in sHLA-G-treated NK cells. sHLA-G up-regulated secretion of (1) CCL22 in CD56bright and CD56dim and (2) CCL2, CCL8, and CXCL2-CXCL3 in CD56dim PB NK cells. Signal transduction experiments showed sHLA-G-mediated down-modulation of Stat5 phosphorylation in PB NK cells. In conclusion, our data delineated novel mechanisms of sHLA-G-mediated inhibition of NK-cell functions.

Manufacturing Natural Killer Cells as Medicinal Products

PubMed Central

Chabannon, Christian; Mfarrej, Bechara; Guia, Sophie; Ugolini, Sophie; Devillier, Raynier; Blaise, Didier; Vivier, Eric; Calmels, Boris

2016-01-01

Natural Killer (NK) cells are innate lymphoid cells (ILC) with cytotoxic and regulatory properties. Their functions are tightly regulated by an array of inhibitory and activating receptors, and their mechanisms of activation strongly differ from antigen recognition in the context of human leukocyte antigen presentation as needed for T-cell activation. NK cells thus offer unique opportunities for new and improved therapeutic manipulation, either in vivo or in vitro, in a variety of human diseases, including cancers. NK cell activity can possibly be modulated in vivo through direct or indirect actions exerted by small molecules or monoclonal antibodies. NK cells can also be adoptively transferred following more or less substantial modifications through cell and gene manufacturing, in order to empower them with new or improved functions and ensure their controlled persistence and activity in the recipient. In the present review, we will focus on the technological and regulatory challenges of NK cell manufacturing and discuss conditions in which these innovative cellular therapies can be brought to the clinic. PMID:27895646

High-efficiency lysis of cervical cancer by allogeneic NK cells derived from umbilical cord progenitors is independent of HLA status.

PubMed

Veluchamy, John P; Heeren, A Marijne; Spanholtz, Jan; van Eendenburg, Jaap D H; Heideman, Daniëlle A M; Kenter, Gemma G; Verheul, Henk M; van der Vliet, Hans J; Jordanova, Ekaterina S; de Gruijl, Tanja D

2017-01-01

Down-regulation of HLA in tumor cells, low numbers and dysfunctionality of NK cells are commonly observed in patients with end-stage cervical cancer. Adoptive transfer of high numbers of cytotoxic NK cells might be a promising treatment approach in this setting. Here, we explored the cytotoxic efficacy on ten cervical cancer cell lines of activated allogeneic NK cells from two sources, i.e., peripheral blood (PBNK) with and without cetuximab (CET), a tumor-specific monoclonal antibody directed against EGFR, or derived from umbilical cord blood (UCB-NK). Whereas CET monotherapy was ineffective against the panel of cervical cancer cell lines, irrespective of their EGFR expression levels and despite their RAS wt status, it significantly enhanced the in vitro cytotoxic efficacy of activated PBNK (P = 0.002). Equally superior cytotoxicity over activated PBNK alone was achieved by UCB-NK (P < 0.001). Both PBNK- and UCB-NK-mediated cytotoxic activity was dependent on the NK-activating receptors natural killer group 2, member D receptor (NKG2D) and DNAX accessory molecule-1 (DNAM-1) (P < 0.05) and unrelated to expression levels of the inhibitory receptors HLA-E and/or HLA-G. Most strikingly, whereas the PBNK's cytotoxic activity was inversely correlated with HLA-ABC levels (P = 0.036), PBNK + CET and UCB-NK cytotoxicity were entirely independent of HLA-ABC expression. In conclusion, this study provides a rationale to initiate a clinical trial for cervical cancer with adoptively transferred allogeneic NK cells, employing either UCB-NK or PBNK + CET for EGFR-expressing tumors. Adoptive transfer of UCB-NK might serve as a generally applicable treatment for cervical cancer, enabled by HLA-, histology- and HPV-independent killing mechanisms.

The effect of mifepristone on the peripheral blood natural killer cell's cytotoxicity and expression of CD94/NKG2A and NKG2D during the implantation phase.

PubMed

Chen, Xiu-Ying; Zhuang, Ya-Ling; Li, Li; Zhang, Wu-Wen; Huang, Li-Li

2010-05-15

To investigate the effect of mifepristone on peripheral blood natural killer cell's (pbNK) cytotoxicity and the expression of the inhibitory receptor CD94/NKG2A and the activated receptor NKG2D on pbNK cells. In vitro study. University hospital and research laboratory. Twenty healthy nonpregnant women. Detected the cytolytic activity of pbNK to K562 target cells; measured the expression of CD94/NKG2A and NKG2D on pbNK. Cytotoxicity of pbNK was detected by Methyl thiazolyl tetrazolium. The expression of CD94/NKG2A and NKG2D receptor on pbNK cells were detected by flow cytometry. The NK cell cytotoxicity and the expression of inhibitory receptor CD94/NKG2A during the proliferative phase (81.71 +/- 11.5, 86.6 +/- 9.0) was significantly higher than the secretory phase (60.16 +/- 19.2, 60.15 +/- 31.0). The NK cells cytotoxicity, after being treated with mifepristone and the expression of inhibitory receptor CD94/NKG2A on pbNK cells treated with 200 nmol/L mifepristone, were significantly increased. Mifepristone had no effect on the expression of activating receptor NKG2D. These data suggest that Mifepristone maybe exert its anti-implantation function by increasing NK cytotoxicity. The increasing NK cytotoxicity of mifepristone is not related to CD94/NKG2A and NKG2D. In the secretory phase down-regulated CD94/NKG2A, NKG2D, and NK cytotoxicity may benefit with embryo implantation. Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.

Requirement and Redundancy of the Src Family Kinases Fyn and Lyn in Perforin-Dependent Killing of Cryptococcus neoformans by NK Cells

PubMed Central

Oykhman, Paul; Timm-McCann, Martina; Xiang, Richard F.; Islam, Anowara; Li, Shu Shun; Stack, Danuta; Huston, Shaunna M.; Ma, Ling Ling

2013-01-01

Natural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungus Cryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase–extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blocked C. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse. PMID:23918783

Natural killer cells regulate T cell immune responses in primary biliary cirrhosis.

PubMed

Shimoda, Shinji; Hisamoto, Satomi; Harada, Kenichi; Iwasaka, Sho; Chong, Yong; Nakamura, Minoru; Bekki, Yuki; Yoshizumi, Tomoharu; Shirabe, Ken; Ikegami, Toru; Maehara, Yoshihiko; He, Xiao-Song; Gershwin, M Eric; Akashi, Koichi

2015-12-01

The hallmark of primary biliary cirrhosis (PBC) is the presence of autoreactive T- and B-cell responses that target biliary epithelial cells (BECs). Biliary cell cytotoxicity is dependent upon initiation of innate immune responses followed by chronic adaptive, as well as bystander, mechanisms. Critical to these mechanisms are interactions between natural killer (NK) cells and BECs. We have taken advantage of the ability to isolate relatively pure viable preparations of liver-derived NK cells, BECs, and endothelial cells, and studied interactions between NK cells and BECs and focused on the mechanisms that activate autoreactive T cells, their dependence on interferon (IFN)-γ, and expression of BEC major histocompatibility complex (MHC) class I and II molecules. Here we show that at a high NK/BEC ratio, NK cells are cytotoxic for autologous BECs, but are not dependent on autoantigen, yet still activate autoreactive CD4(+) T cells in the presence of antigen presenting cells. In contrast, at a low NK/BEC ratio, BECs are not lysed, but IFN-γ production is induced, which facilitates expression of MHC class I and II molecules on BEC and protects them from lysis upon subsequent exposure to autoreactive NK cells. Furthermore, IFN-γ secreted from NK cells after exposure to autologous BECs is essential for this protective function and enables autoreactive CD4(+) T cells to become cytopathic. NK cell-mediated innate immune responses are likely critical at the initial stage of PBC, but also facilitate and maintain the chronic cytopathic effect of autoantigen-specific T cells, essential for progression of disease. © 2015 by the American Association for the Study of Liver Diseases.

Combined Stimulation with Interleukin-18 and Interleukin-12 Potently Induces Interleukin-8 Production by Natural Killer Cells.

PubMed

Poznanski, Sophie M; Lee, Amanda J; Nham, Tina; Lusty, Evan; Larché, Margaret J; Lee, Dean A; Ashkar, Ali A

2017-01-01

The combination of interleukin (IL)-18 and IL-12 (IL-18+IL-12) potently stimulates natural killer (NK) cells, triggering an innate immune response to infections and cancers. Strategies exploiting the effects of IL-18+IL-12 have shown promise for cancer immunotherapy. However, studies have primarily characterized the NK cell response to IL-18+IL-12 in terms of interferon (IFN)-γ production, with little focus on other cytokines produced. IL-8 plays a critical role in activating and recruiting immune cells, but it also has tumor-promoting functions. IL-8 is classically produced by regulatory NK cells; however, cytotoxic NK cells do not typically produce IL-8. In this study, we uncover that stimulation with IL-18+IL-12 induces high levels of IL-8 production by ex vivo expanded and freshly isolated NK cells and NK cells in peripheral blood mononuclear cells. We further report that tumor necrosis factor (TNF)-α, produced by NK cells following IL-18+IL-12 stimulation, regulates IL-8 production. The IL-8 produced is in turn required for maximal IFN-γ and TNF-α production. These findings may have important implications for the immune response to infections and cancer immunotherapies. This study broadens our understanding of NK cell function and IL-18+IL-12 synergy by uncovering an unprecedented ability of IL-18+IL-12-activated peripheral blood NK cells to produce elevated levels of IL-8 and identifying the requirement for intermediates induced by IL-18+IL-12 for maximal cytokine production following stimulation. © 2017 S. Karger AG, Basel.

Genome-wide siRNA screen reveals a new cellular partner of NK cell receptor KIR2DL4: heparan sulfate directly modulates KIR2DL4-mediated responses

PubMed Central

Brusilovsky, Michael; Cordoba, Moti; Rosental, Benyamin; Hershkovitz, Oren; Andrake, Mark D.; Pecherskaya, Anna; Einarson, Margret B.; Zhou, Yan; Braiman, Alex

2013-01-01

KIR2DL4 (CD158d) is a distinct member of the killer cell Ig-like receptor (KIR) family in human NK cells that can induce cytokine production and cytolytic activity in resting NK cells. Soluble HLA-G, normally expressed only by fetal-derived trophoblast cells, was reported to be a ligand for KIR2DL4; however, KIR2DL4 expression is not restricted to the placenta and can be found in CD56high subset of peripheral blood NK cells. We demonstrated that KIR2DL4 can interact with alternative ligand(s), expressed by cells of epithelial or fibroblast origin. A genome-wide high-throughput siRNA screen revealed that KIR2DL4 recognition of cells surface ligand(s) is directly regulated by heparan sulfate (HS) glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1). KIR2DL4 was found to directly interact with HS/heparin, and the D0-domain of KIR2DL4 was essential for this interaction. Accordingly, exogenous HS/heparin can regulate cytokine production by KIR2DL4-expressing NK cells and HEK293T cells (HEK293T-2DL4) and induces differential localization of KIR2DL4 to rab5+ and rab7+ endosomes, thus leading to down-regulation of cytokine production and degradation of the receptor. Furthermore, we showed that intimate interaction of syndecan-4 (SDC4) HS Proteo-Glycan (HSPG) and KIR2DL4 directly affects receptor endocytosis and membrane trafficking. PMID:24127555

Acute virus control mediated by licensed NK cells sets primary CD8+ T cell dependence on CD27 costimulation1,2,3

PubMed Central

Teoh, Jeffrey J.; Gamache, Awndre E.; Gillespie, Alyssa L.; Stadnisky, Michael D.; Yagita, Hideo; Bullock, Timothy N.J.; Brown, Michael G.

2016-01-01

Natural killer (NK) cells represent a critical first-line of immune defense against a bevy of viral pathogens, and infection can provoke them to mediate both supportive and suppressive effects on virus-specific adaptive immunity. In mice expressing MHC I Dk, a major MCMV resistance factor and self-ligand of the inhibitory Ly49G2 (G2) receptor, licensed G2+ NK cells provide essential host resistance against murine (M)CMV infection. Additionally G2+ NK cell responses to MCMV increase the rate and extent of dendritic cell (DC) recovery, as well as early priming of CD8+ T-cell effectors in response to MCMV. However, relatively little is known about the NK-cell effect on co-stimulatory ligand patterns displayed by DCs, or ensuing effector and memory T-cell responses. Here we found that CD27-dependent CD8+ T-cell priming and differentiation is shaped by the efficiency of NK responses to virus infection. Surprisingly, differences in specific NK responses to MCMV in Dk-disparate mice failed to distinguish early DC co-stimulatory patterns. Nonetheless, while CD27 deficiency did not impede licensed NK-mediated resistance, both CD70 and CD27 were required to efficiently prime and regulate effector CD8+ T-cell differentiation in response to MCMV, which eventually resulted in biased memory T-cell precursor formation in Dk mice. In contrast, CD8+ T-cells accrued more slowly in non-Dk mice, and eventually differentiated into terminal effector cells regardless of CD27 stimulation. Disparity in this requirement for CD27 signaling indicates that specific virus control mediated by NK cells can shape DC co-stimulatory signals needed to prime CD8+ T cells and eventual T-cell fate decisions. PMID:27798162

MICA/B expression is inhibited by unfolded protein response and associated with poor prognosis in human hepatocellular carcinoma.

PubMed

Fang, Liang; Gong, Jiuyu; Wang, Ying; Liu, Rongrong; Li, Zengshan; Wang, Zhe; Zhang, Yun; Zhang, Chunmei; Song, Chaojun; Yang, Angang; Ting, Jenny P-Y; Jin, Boquan; Chen, Lihua

2014-09-18

MICA/B are major ligands for NK cell activating receptor NKG2D and previous studies showed that the serum level of soluble MICA (sMICA) is an independent prognostic factor for advanced human hepatocellular carcinoma. However, the correlation between cellular MICA/B expression pattern and human hepatocellular carcinoma progression has not been well explored. The unfolded protein response is one of the main causes of resistance to chemotherapy and radiotherapy in tumor cells. However, whether the UPR in HCC could regulate the expression levels of MICA/B and affect the sensitivity of HCC cells to NK cell cytolysis has not been established yet. MICA/B expression pattern was evaluated by immunohistochemistry and Kaplan-Meier survival analysis was done to explore the relationship between MICA/B expression level and patient survival. The protein and mRNA expression levels of MICA/B in SMMC7721 and HepG2 cells treated by tunicamycin were evaluated by flow cytometry, Western Blot and RT-PCR. The cytotoxicity analysis was performed with the CytoTox 96 Non-Radioactive LDH Cytotoxicity Assay. MICA/B was highly expressed in human hepatocellular carcinoma and the expression level was significantly and negatively associated with tumor-node metastasis (TNM) stages. Patients with low level of MICA/B expression showed a trend of shorter survival time. The unfolded protein response (UPR) downregulated the expression of MICA/B. This decreased protein expression occurred via post-transcriptional regulation and was associated with proteasomal degradation. Moreover, decreased expression level of MICA/B led to the attenuated sensitivity of human HCC to NK cell cytotoxicity. These new findings of the connection of MICA/B, UPR and NK cells may represent a new concrete theory of NK cell regulation in HCC, and suggest that targeting this novel NK cell-associated immune evasion pathway may be meaningful in treating patients with HCC.

Cytotoxic potential of decidual NK cells and CD8+ T cells awakened by infections.

PubMed

Crespo, Ângela C; van der Zwan, Anita; Ramalho-Santos, João; Strominger, Jack L; Tilburgs, Tamara

2017-02-01

To establish a healthy pregnancy the maternal immune system must tolerate fetal allo-antigens, yet remain competent to respond to infections. The ability of decidual NK cells (dNK) to promote migration of fetal extravillous trophoblasts (EVT) and placental growth as well as the capacity of EVT to promote immune tolerance are topics of high interest and extensive research. However, the problem of how dNK and decidual CD8+ T cells (CD8+ dT) provide immunity to infections of the placenta and the mechanisms that regulate their cytolytic function has thus far largely been ignored. Fetal EVT are the most invasive cells of the placenta and directly interact with maternal decidual immune cells at this maternal-fetal interface. Besides the expression of non-polymorphic HLA-E and HLA-G molecules that are associated with immune tolerance, EVT also express highly polymorphic HLA-C molecules that can serve as targets for maternal dNK and CD8+ dT responses. HLA-C expression by EVT has a dual role as the main molecule to which immune tolerance needs to be established and as the only molecule that can present pathogen-derived peptides and provide protective immunity when EVT are infected. The focus of this review is to address the regulation of cytotoxicity of dNK and CD8+ dT, which is essential for maternal-fetal immune tolerance as well as recent evidence that both cell types can provide immunity to infections at the maternal-fetal interface. A particular emphasis is given to the role of HLA-C expressed by EVT and its capacity to elicit dNK and CD8+ dT responses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Decidualization and angiogenesis in early pregnancy: unravelling the functions of DC and NK cells.

PubMed

Blois, Sandra M; Klapp, Burghard F; Barrientos, Gabriela

2011-03-01

Differentiation of endometrial stromal cells and formation of new maternal blood vessels at the time of embryo implantation are critical for the establishment and maintenance of gestation. The regulatory functions of decidual leukocytes during early pregnancy, particularly dendritic cells (DC) and NK cells, may be important not only for the generation of maternal immunological tolerance but also in the regulation of stromal cell differentiation and the vascular responses associated with the implantation process. However, the specific contributions of DC and NK cells during implantation are still difficult to dissect mainly due to reciprocal regulatory interactions established between them within the decidualizing microenvironment. The present review article discusses current evidence on the regulatory pathways driving decidualization in mice, suggesting that NK cells promote uterine vascular modifications that assist decidual growth but DC directly control stromal cell proliferation, angiogenesis and the homing and maturation of NK cell precursors in the pregnant uterus. Thus, successful implantation appears to result from an interplay between cellular components of the decidualizing endometrium involving immunoregulatory and pro-angiogenic functions of DC and NK cells. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides

PubMed Central

Schafer, Jamie L.; Ries, Moritz; Guha, Natasha; Connole, Michelle; Colantonio, Arnaud D.; Wiertz, Emmanuel J.; Wilson, Nancy A.; Kaur, Amitinder; Evans, David T.

2015-01-01

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05+ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05+ NK cells, including three immunodominant CD8+ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05+ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002+ CD4+ lymphocytes co-cultured with Mamu-KIR3DL05+ NK cells than with Mamu-KIR3DL05- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses. PMID:26333068

IL-12-dependent inducible expression of the CD94/NKG2A inhibitory receptor regulates CD94/NKG2C+ NK cell function.

PubMed

Sáez-Borderías, Andrea; Romo, Neus; Magri, Giuliana; Gumá, Mónica; Angulo, Ana; López-Botet, Miguel

2009-01-15

The inhibitory CD94/NKG2A and activating CD94/NKG2C killer lectin-like receptors specific for HLA-E have been reported to be selectively expressed by discrete NK and T cell subsets. In the present study, minor proportions of NK and T cells coexpressing both CD94/NKG2A and CD94/NKG2C were found in fresh peripheral blood from adult blood donors. Moreover, CD94/NKG2A surface expression was transiently detected upon in vitro stimulation of CD94/NKG2C+ NK cells in the presence of irradiated allogeneic PBMC or rIL-12. A similar effect was observed upon coculture of NKG2C+ NK clones with human CMV-infected autologous dendritic cell cultures, and it was prevented by an anti-IL-12 mAb. NKG2A inhibited the cytolytic activity of NKG2C+ NK clones upon engagement either by a specific mAb or upon interaction with a transfectant of the HLA class I-deficient 721.221 cell line expressing HLA-E. These data indicate that beyond its constitutive expression by an NK cell subset, NKG2A may be also transiently displayed by CD94/NKG2C+ NK cells under the influence of IL-12, providing a potential negative regulatory feedback mechanism.

The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection.

PubMed

Rajalingam, Raja

2016-01-01

Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.

Hypoxic tumor-derived microvesicles negatively regulate NK cell function by a mechanism involving TGF-β and miR23a transfer.

PubMed

Berchem, Guy; Noman, Muhammad Zaeem; Bosseler, Manon; Paggetti, Jerome; Baconnais, Sonia; Le Cam, Eric; Nanbakhsh, Arash; Moussay, Etienne; Mami-Chouaib, Fathia; Janji, Bassam; Chouaib, Salem

2016-04-01

Tumor-derived microvesicles (TD-MVs) are key mediators which are shed by cancer cells and can sensitize neighboring cells in the tumor microenvironment. TD-MVs are extracellular vesicles composed of exosomes and MVs and promote cancer invasion and metastasis. Intratumoral hypoxia is an integral component of all solid tumors. The relationship between hypoxic tumor-shed MVs and NK-mediated cytotoxicity remains unknown. In this paper, we reported that MVs derived from hypoxic tumor cells qualitatively differ from those derived from normoxic tumor cells. Using multiple tumor models, we showed that hypoxic MVs inhibit more NK cell function as compared to normoxic MVs. Hypoxic TD-MVs package two immunosuppressive factors involved in the impairment of natural killer (NK) cell cytotoxicity against different tumor cells in vitro and in vivo . We showed that following their uptake by NK cells, hypoxic TD-MVs transfer TGF-β1 to NK cells, decreasing the cell surface expression of the activating receptor NKG2D, thereby inhibiting NK cell function. MicroRNA profiling revealed the presence of high levels of miR-210 and miR-23a in hypoxic TD-MVs. We demonstrated that miR-23a in hypoxic TD-MVs operates as an additional immunomosuppressive factor, since it directly targets the expression of CD107a in NK cells. To our knowledge, this is the first study to show that hypoxic tumor cells by secreting MVs can educate NK cells and decrease their antitumor immune response. This study highlights the existence of a novel mechanism of immune suppression mediated by hypoxic TD-MVs and further improves our understanding of the immunosuppressive mechanisms prevailing in the hypoxic tumor microenvironment.

NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors.

PubMed

Anton, Olga M; Vielkind, Susina; Peterson, Mary E; Tagaya, Yutaka; Long, Eric O

2015-11-15

IL-15 bound to the IL-15Rα-chain (IL-15Rα) is presented in trans to cells bearing the IL-2Rβ-chain and common γ-chain. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor-ligand interactions. In this study, human NK cells were tested for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Rα. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIR-HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to downregulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A(+) cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Coengagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across, inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15-dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis. Copyright © 2015 by The American Association of Immunologists, Inc.

Scorpion venom activates natural killer cells in hepatocellular carcinoma via the NKG2D-MICA pathway.

PubMed

Chen, Han; Zhidan, Wang; Xia, Ren; Zhaoxia, Wang; Qing, Jia; Qiang, Guo; Haipeng, Yin; Hengxiao, Wang

2016-06-01

Previous studies have demonstrated that polypeptides extracted from scorpion venom (PESV) inhibited cell proliferation in several tumors, however, the effect on dysfunctional and exhausted natural killer cells which contribute to tumor escape from immune surveillance remain to be elucidated. In this study, we determined the effect of PESV on NK infiltration into H22 cells orthotopic transplantation tumors and on the expression of MHC class I chain-related proteins A (MICA) in HepG2 cells. We found that tumor growth in mice was significantly inhibited by PESV and the survival time of tumor-bearing mice treated with PESV was significantly prolonged. Moreover, levels of tumor-infiltrating NK cells, NKG2D protein, perforin and granzyme B mRNA were significantly increased in the group treated with PESV compared with the tumor-bearing control group. In addition, In addition, up-regulation of MICA by PESV enhances the susceptibility of HepG2 cells to NK lysis in vitro. These results indicate that the inhibitory effects of PESV on hepatic carcinoma are likely mediated by up-regulation of NK cell activity via the MICA-NKG2D pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

Human NK Cell Subset Functions Are Differentially Affected by Adipokines

PubMed Central

Huebner, Lena; Engeli, Stefan; Wrann, Christiane D.; Goudeva, Lilia; Laue, Tobias; Kielstein, Heike

2013-01-01

Background Obesity is a risk factor for various types of infectious diseases and cancer. The increase in adipose tissue causes alterations in both adipogenesis and the production of adipocyte-secreted proteins (adipokines). Since natural killer (NK) cells are the host’s primary defense against virus-infected and tumor cells, we investigated how adipocyte-conditioned medium (ACM) affects functions of two distinct human NK cell subsets. Methods Isolated human peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of human and murine ACM harvested on two different days during adipogenesis and analyzed by fluorescent-activated cell sorting (FACS). Results FACS analyses showed that the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme A (GzmA) and interferon (IFN)-γ in NK cells was regulated in a subset-specific manner. ACM treatment altered IFN-γ expression in CD56dim NK cells. The production of GzmA in CD56bright NK cells was differentially affected by the distinct adipokine compositions harvested at different states of adipogenesis. Comparison of the treatment with either human or murine ACM revealed that adipokine-induced effects on NK cell expression of the leptin receptor (Ob-R), TRAIL and IFN-γ were species-specific. Conclusion Considering the growing prevalence of obesity and the various disorders related to it, the present study provides further insights into the roles human NK cell subsets play in the obesity-associated state of chronic low-grade inflammation. PMID:24098717

Activation of natural killer cells by hepatitis C virus particles in vitro.

PubMed

Farag, M M S; Weigand, K; Encke, J; Momburg, F

2011-09-01

Little is known about the ability of hepatitis C virus (HCV) to alter early innate immune responses in infected patients. Previous studies have shown that natural killer (NK) cells are functionally impaired after interaction of recombinant HCV glycoprotein E2 with the co-stimulatory CD81 molecule in vitro; however, the functional consequences of a prolonged contact of NK cells with HCV particles have remained unclear. We have examined the phenotypes of purified, interleukin-2-activated NK cells from healthy donors and HCV genotype 1b patients after culture for 5 days with HCV pseudoparticles (HCVpp) and serum samples containing HCV genotype 1b. NK cells from healthy donors and chronic HCV patients were found to up-regulate receptors associated with activation (NKp46, NKp44, NKp30, NKG2D), while NK receptors from the killer cell immunoglobulin-like receptor family (KIR/CD158), predominantly having an inhibitory function, were significantly down-modulated after culture in the presence of HCV particles compared with control cultures of NK cells. HCV-infected sera and HCVpp elicited significantly higher secretion of the NK effector lymphokines interferon-γ and tumour necrosis factor-α. Furthermore, HCV stimulated the cytotoxic potential of NK cells from normal donors and patients. The enhanced activation of NK cells after prolonged culture with HCVpp or HCV-containing sera for 5 days suggests that these innate effector cells may play an important role in viral control during early phases of HCV infection. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME).

PubMed

Petty, Robert D; McCarthy, Neil E; Le Dieu, Rifca; Kerr, Jonathan R

2016-01-01

Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients. miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets. Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology. This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function.

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME)

PubMed Central

Petty, Robert D.; McCarthy, Neil E.; Le Dieu, Rifca; Kerr, Jonathan R.

2016-01-01

Background Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients. Methods miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets. Results Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology. Conclusion This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function. PMID:26967895

Saliva exosomes from pancreatic tumor-bearing mice modulate NK cell phenotype and antitumor cytotoxicity.

PubMed

Katsiougiannis, Stergios; Chia, David; Kim, Yong; Singh, Ram P; Wong, David T W

2017-03-01

Tumor exosomes are emerging as antitumor immunity regulators; however, their effects on secondary exosome secretion by distal organs have not been explored. We have previously demonstrated that suppression of exosomes at the distal tumor site of pancreatic ductal adenocarcinoma (PDAC) ablated the development of salivary biomarker profile. Here, we explore the function of salivary exosomes from tumor-bearing mice in immune surveillance. We provide evidence that salivary exosomes from mice with PDAC exhibit a suppressive effect that results in reduced tumor-killing capacity by NK cells. Salivary exosomes from mice with PDAC where pancreatic tumors were engineered to suppress exosome biogenesis failed to suppress NK cell cytotoxic potential against tumor cells, as opposed to salivary exosomes from mice with PDAC with normal tumor exosome biogenesis. These results reveal an important and previously unknown mechanism of antitumor immune regulation and provide new insights into our understanding of the alterations of this biofluid during tumor development.-Katsiougiannis, S., Chia, D., Kim, Y., Singh, R. P., Wong, D. T. W. Saliva exosomes from pancreatic tumor-bearing mice modulate NK cell phenotype and antitumor cytotoxicity. © FASEB.

Neurokinin 1 Receptor Mediates Membrane Blebbing and Sheer Stress-Induced Microparticle Formation in HEK293 Cells

PubMed Central

Chen, Panpan; Douglas, Steven D.; Meshki, John; Tuluc, Florin

2012-01-01

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2–10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing. PMID:23024816

Neurokinin 1 receptor mediates membrane blebbing and sheer stress-induced microparticle formation in HEK293 cells.

PubMed

Chen, Panpan; Douglas, Steven D; Meshki, John; Tuluc, Florin

2012-01-01

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2-10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.

COX-2 plays a role in angiogenic DBA(+) uNK cell subsets activation and pregnancy protection in LPS-exposed mice.

PubMed

Zavan, Bruno; De Almeida, Eliana Martins; Salles, Évila da Silva Lopes; do Amarante-Paffaro, Andréa Mollica; Paffaro, Valdemar Antonio

2016-08-01

Although uterine Natural Killer (uNK) cells have cytoplasmic granules rich in perforin and granzymes, these cells do not degranulate in normal pregnancy. DBA lectin(+) uNK cells produce angiogenic factors which stimulate remodeling of uterine arterioles to increase blood flow within the growing feto-placental unit. We sought to investigate the importance of COX-2 on mouse pregnancy inoculated with Gram-negative bacteria Lipopolysaccharide (LPS) by treating with a selective COX-2 inhibitor (nimesulide). We have combined histochemical, immunohistochemical, stereological, morphometric, behavioral, and litter analyses to investigate mouse pregnancy inoculated with LPS with or without pre-treatment with nimesulide 30 min before LPS injections, focusing on DBA(+) uNK cell response and viability of the pregnancy. LPS caused sickness behavior, an immature DBA(+) uNK influx, decreased mature DBA(+) uNK cell numbers, and triggered a new DBA(low) uNK appearance. These effects of LPS, except the sickness behavior, were prevented by nimesulide. COX-2 inhibition also prevented the down-regulation of uNK perforin and spiral arteriole α-actin expression stimulated by LPS. While the litter size from Nimesulide + LPS-treated mothers was significantly smaller compared to those from LPS-treated group, nimesulide alone showed no effect on the offspring. Collectively, our data indicate that COX-2 changes angiogenic DBA(+) uNK cells in order to protect mouse pregnancy after LPS injection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cultured Mycelium Cordyceps sinensis allevi¬ates CCl4-induced liver inflammation and fibrosis in mice by activating hepatic natural killer cells.

PubMed

Peng, Yuan; Huang, Kai; Shen, Li; Tao, Yan-yan; Liu, Cheng-hai

2016-02-01

Recent evidence shows that cultured mycelium Cordyceps sinensis (CMCS) effectively protects against liver fibrosis in mice. Here, we investigated whether the anti-fibrotic action of CMCS was related to its regulation of the activity of hepatic natural killer (NK) cells in CCl4-treated mice. C57BL/6 mice were injected with 10% CCl4 (2 mL/kg, ip) 3 times per week for 4 weeks, and received CMCS (120 mg·kg(-1)·d(-1), ig) during this period. In another part of experiments, the mice were also injected with an NK cell-deleting antibody ASGM-1 (20 μg, ip) 5 times in the first 3 weeks. After the mice were sacrificed, serum liver function, and liver inflammation, hydroxyproline content and collagen deposition were assessed. The numbers of hepatic NK cells and expression of NKG2D (activation receptor of NK cells) on isolated liver lymphocytes were analyzed using flow cytometry. Desmin expression and cell apoptosis in liver tissues were studied using desmin staining and TUNEL assay, respectively. The levels of α-SMA, TGF-β, RAE-1δ and RAE-1ε in liver tissues were determined by RT-qPCR. In CCl4-treated mice, CMCS administration significantly improved liver function, attenuated liver inflammation and fibrosis, and increased the numbers of hepatic NK cells and expression level of NKG2D on hepatic NK cells. Furthermore, CMCS administration significantly decreased desmin expression in liver tissues, and increased TUNEL staining adjacent to hepatic stellate cells. Injection with NK cell-deleting ASGM-1 not only diminished the numbers of hepatic NK cells, but also greatly accelerated liver inflammation and fibrosis in CCl4-treated mice. In CCl4-treated mice with NK cell depletion, CMCS administration decelerated the rate of liver fibrosis development, and mildly upregulated the numbers of hepatic NK cells but without changing NKG2D expression. CMCS alleviates CCl4-induced liver inflammation and fibrosis via promoting activation of hepatic NK cells. CMCS partially reverses ASGM-1-induced depletion of hepatic NK cells.

CXCR3/CXCL10 Axis Regulates Neutrophil-NK Cell Cross-Talk Determining the Severity of Experimental Osteoarthritis.

PubMed

Benigni, Giorgia; Dimitrova, Petya; Antonangeli, Fabrizio; Sanseviero, Emilio; Milanova, Viktoriya; Blom, Arjen; van Lent, Peter; Morrone, Stefania; Santoni, Angela; Bernardini, Giovanni

2017-03-01

Several immune cell populations are involved in cartilage damage, bone erosion, and resorption processes during osteoarthritis. The purpose of this study was to investigate the role of NK cells in the pathogenesis of experimental osteoarthritis and whether and how neutrophils can regulate their synovial localization in the disease. Experimental osteoarthritis was elicited by intra-articular injection of collagenase in wild type and Cxcr3 -/- 8-wk old mice. To follow osteoarthritis progression, cartilage damage, synovial thickening, and osteophyte formation were measured histologically. To characterize the inflammatory cells involved in osteoarthritis, synovial fluid was collected early after disease induction, and the cellular and cytokine content were quantified by flow cytometry and ELISA, respectively. We found that NK cells and neutrophils are among the first cells that accumulate in the synovium during osteoarthritis, both exerting a pathogenic role. Moreover, we uncovered a crucial role of the CXCL10/CXCR3 axis, with CXCL10 increasing in synovial fluids after injury and Cxcr3 -/- mice being protected from disease development. Finally, in vivo depletion experiments showed that neutrophils are involved in an NK cell increase in the synovium, possibly by expressing CXCL10 in inflamed joints. Thus, neutrophils and NK cells act as important disease-promoting immune cells in experimental osteoarthritis and their functional interaction is promoted by the CXCL10/CXCR3 axis. Copyright © 2017 by The American Association of Immunologists, Inc.

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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Endocytosis as a mechanism of regulating natural killer cell function: unique endocytic and trafficking pathway for CD94/NKG2A.

PubMed

Peruzzi, Giovanna; Masilamani, Madhan; Borrego, Francisco; Coligan, John E

2009-01-01

Natural killer (NK) cells are lymphocytes generally recognized as sentinels of the innate immune system due to their inherent capacity to deal with diseased (stressed) cells, including malignant and infected. This ability to recognize many potentially pathogenic situations is due to the expression of a diverse panel of activation receptors. Because NK cell activation triggers an aggressive inflammatory response, it is important to have a means of throttling this response. Hence, NK cells also express a panel of inhibitory receptors that recognize ligands expressed by "normal" cells. Little or nothing is known about the endocytosis and trafficking of NK cell receptors, which are of great relevance to understanding how NK cells maintain the appropriate balance of activating and inhibitory receptors on their cell surface. In this review, we focus on the ITIM-containing inhibitory receptor CD94/NKG2A showing that it is endocytosed by a previously undescribed macropinocytic-like process that may be related to the maintenance of its surface expression.

The aryl hydrocarbon receptor is required for the maintenance of liver-resident natural killer cells

PubMed Central

2016-01-01

A tissue-resident population of natural killer cells (NK cells) in the liver has recently been described to have the unique capacity to confer immunological memory in the form of hapten-specific contact hypersensitivity independent of T and B cells. Factors regulating the development and maintenance of these liver-resident NK cells are poorly understood. The aryl hydrocarbon receptor (AhR) is a transcription factor modulated by exogenous and endogenous ligands that is important in the homeostasis of immune cells at barrier sites, such as the skin and gut. In this study, we show that liver-resident NK (NK1.1+CD3−) cells, defined as CD49a+TRAIL+CXCR6+DX5− cells in the mouse liver, constitutively express AhR. In AhR−/− mice, there is a significant reduction in the proportion and absolute number of these cells, which results from a cell-intrinsic dependence on AhR. This deficiency in liver-resident NK cells appears to be the result of higher turnover and increased susceptibility to cytokine-induced cell death. Finally, we show that this deficiency has functional implications in vivo. Upon hapten exposure, AhR−/− mice are not able to mount an NK cell memory response to hapten rechallenge. Together, these data demonstrate the requirement of AhR for the maintenance of CD49a+TRAIL+CXCR6+DX5− liver-resident NK cells and their hapten memory function. PMID:27670593

Functional role of human NK cell receptor 2B4 (CD244) isoforms.

PubMed

Mathew, Stephen O; Rao, Krithi K; Kim, Jong R; Bambard, Nowland D; Mathew, Porunelloor A

2009-06-01

2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM/CD150), is expressed on all NK cells, a subpopulation of T cells, monocytes and basophils. Human NK cells express two isoforms of 2B4, h2B4-A and h2B4-B that differ in a small portion of the extracellular domain. In the present investigation, we have studied the functions of h2B4-A and h2B4-B. Our study demonstrated that these two isoforms differ in their binding affinity for CD48, which results in differential cytotoxic activity as well as intracellular calcium release by NK cells upon target cell recognition. Analysis of the predicted 3-D structure of the two isoforms showed conformational differences that could account for their differences in binding affinity to CD48. h2B4-A was able to mediate natural cytotoxicity against CD48-expressing K562 target cells and induce intracellular calcium release, whereas h2B4-B showed no effects. NK-92MI, U937, THP-1, KU812, primary monocytes, basophils and NK cells showed expression of both h2B4-A and h2B4-B whereas YT and IL-2-activated NK cells did not show any h2B4-B expression. Stimulation of NK cells through 2B4 resulted in decreased mRNA levels of both h2B4-A and h2B4-B indicating that down-regulation of 2B4 isoforms may be an important factor in controlling NK cell activation during immune responses.

The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection

PubMed Central

Rajalingam, Raja

2016-01-01

Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR–HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants. PMID:28066408

Regulation of adaptive NK cells and CD8 T cells by HLA-C correlates with allogeneic hematopoietic cell transplantation and with CMV reactivation1

PubMed Central

Horowitz, Amir; Guethlein, Lisbeth A.; Nemat-Gorgani, Neda; Norman, Paul J.; Cooley, Sarah; Miller, Jeffrey S.; Parham, Peter

2015-01-01

Mass cytometry was used to investigate the effect of CMV reactivation on lymphocyte reconstitution in hematopoietic cell transplant patients. For eight transplant recipients, four CMV negative and four CMV positive, we studied peripheral blood mononuclear cells (PBMC) obtained six months after unrelated donor hematopoietic cell transplantation (HCT). Forty cell-surface markers, distinguishing all major leukocyte populations in PBMC, were analyzed by mass cytometry. These included 34 NK cell markers. Compared to healthy controls, transplant recipients had higher HLA-C expression on CD56−CD16+ NK cells, B cells, CD33bright myeloid cells and CD4CD8 T cells. The increase in HLA-C expression was greater for CMV-positive HCT recipients than CMV negative recipients. Present in CMV-positive HCT recipients, but not in CMV-negative HCT recipients or controls, is a population of KIR-expressing CD8 T cells not previously described. These CD8 T cells co-express CD56, CD57 and NKG2C. The HCT recipients also have a population of CD57+NKG2A+ NK cells that preferentially express KIR2DL1. An inverse correlation was observed between the frequencies of CD57+NKG2C+ NK cells and CD57+NKG2A+ NK cells. Although CD57+NKG2A+ NK cells are less abundant in CMV-positive recipients, their phenotype is of a more activated cell than the CD57+NKG2A+ NK cells of controls and CMV-negative HCT recipients. These data demonstrate that HCT and CMV reactivation are associated with an increased expression of HLA-C. This could influence NK cell education during lymphocyte reconstitution. The increased inhibitory KIR expression by proliferating CMV-specific CD8 T cells suggests regulatory interactions between HLA-C and KIR might promote GVL effects following transplantation. PMID:26416275

Regulation of the Prostate Cancer Tumor Microenvironment

DTIC Science & Technology

2014-04-01

cells at 24 and 30 weeks of age already reveal increased infiltration of macrophage lineage cells , decreased CD8 T lymphocytes , NK cells , as...NF-κB) and interferon regulatory factors (IRFs), which may mediate the development of cytotoxic T lymphocytes (CTLs) and dendritic cell (DC...tumor infiltration of CD11b+ cells , decreased CD8+ T lymphocytes , and NK cells . • We have shown that absence of MyD88 while leads to

Effects of Butyltins (BTs) on Mitogen-Activated-Protein Kinase Kinase Kinase (MAP3K) and Ras Activity in Human Natural Killer Cells

PubMed Central

Celada, Lindsay J.; Whalen, Margaret M.

2013-01-01

Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT), diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally-infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to the activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 minutes of TBT exposure and the MAP3K, ASK1, after one hour exposures to TBT. In addition, our results suggest that both TBT and DBT are impacting the regulation of c-Raf. PMID:24038145

NK cells modulate the cytotoxic activity generated by Mycobacterium leprae-hsp65 in leprosy patients: role of IL-18 and IL-13

PubMed Central

DE LA BARRERA, S; FINIASZ, M; FINK, S; ILARREGUI, J; ALEMÁN, M; OLIVARES, L; FRANCO, M C; PIZZARIELLO, G; DEL CARMEN SASIAIN, M

2004-01-01

Protection against intracellular pathogens such as Mycobacterium leprae is critically dependent on the function of NK cells at early stages of the immune response and on Th1 cells at later stages. In the present report we evaluated the role of IL-18 and IL-13, two cytokines that can influence NK cell activity, in the generation of M. leprae-derived hsp65-cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) of leprosy patients. We demonstrated that IL-18 modulates hsp65-induced CTL generation and collaborates with IL-12 for this effect. In paucibacillary (PB) patients and normal controls (N) depletion of NK cells reduces the cytolytic activity. Under these conditions, IL-12 cannot up-regulate this CTL generation, while, in contrast, IL-18 increases the cytotoxic activity both in the presence or absence of NK cells. IL-13 down-regulates the hsp65-induced CTL generation and counteracts the positive effect of IL-18. The negative effect of IL-13 is observed in the early stages of the response, suggesting that this cytokine affects IFNγ production by NK cells. mRNA coding for IFNγ is induced by IL-18 and reduced in the presence of IL-13, when PBMC from N or PB patients are stimulated with hsp65. Neutralization of IL-13 in PBMC from multibacillary (MB) leprosy patients induces the production of IFNγ protein by lymphocytes. A modulatory role on the generation of hsp65 induced CTL is demonstrated for IL-18 and IL-13 and this effect takes place through the production of IFNγ. PMID:14678270

NK Depletion Results in Increased CCL22 Secretion and Treg Levels in Lewis Lung Carcinoma via the Accumulation of CCL22-secreting CD11b+CD11c+ Cells

PubMed Central

W., Mailloux, Adam; A., Clark, Anna-Maria; Rita, I. Young, M.

2010-01-01

Tumor induced immune suppression involves the accumulation of suppressive infiltrates in the tumor microenvironment such as regulatory T-cells (Tregs). Previous studies demonstrated that NK-dependant increases in CCL22 secretion selectively recruit Tregs toward murine lungs bearing Lewis Lung Carcinoma (LLC). In order to extend the in vitro studies, the present studies utilized in vivo depletion of NK cells to ascertain the contribution of NK-derived CCL22 toward total CCL22 and subsequent Treg levels in both normal and LLC-bearing lungs. However, NK depletion had the unexpected effect of increasing both CCL22 secretion and Treg levels in the lungs of NK-depleted LLC-bearing mice. This was concurrent with an increase in tumor burden. Flow cytometry and a series of both immunomagnetic and FACS isolations were used to identify the CCL22-producing cellular fractions in LLC-bearing lungs. A novel CD11b+CD11c+ cell population was identified that accumulates in large numbers in NK-depleted LLC-bearing lung tissue. These CD11b+CD11c+ cells secreted large amounts of CCL22 that may overcompensate for the loss of NK-derived CCL22 in the lungs of NK-depleted LLC-bearing mice. Taken together these data suggest that NK cells play both a positive and negative role in the regulation of CCL22 secretion and, in turn, the recruitment of Tregs toward LLC-bearing lungs. PMID:20198623

Expression of the neurokinin type 1 receptor in the human colon.

PubMed

Boutaghou-Cherid, Hikma; Porcher, Christophe; Liberge, Martine; Jule, Yvon; Bunnett, Nigel W; Christen, Marie-Odile

2006-01-30

The distribution of the neurokinin type 1 receptor (NK1r) in human intestine, mapped in a few immunohistochemical investigations in the antrum and the duodenum, is comparable to that widely studied in rodents. Importantly, despite pharmacological evidence of their presence in mammalian intestinal muscle, their immunohistochemical visualization in smooth muscle cells remains to be determined in human digestive tract. In the present work, we studied the distribution of NK1r in the human colon, with a particular view to visualize their expression in muscle cells. With this aim, part of colonic segments were incubated with nicardipine and TTX in order to induce accumulation of the NK1r on cell membrane. NK1r were visualized by using immunohistochemistry combined with fluorescence and confocal microscopy. Without incubation, NK1r-IR was clearly observed on the membrane and the cytoplasm of myenteric and submucous neurons and interstitial cells of Cajal, but could not be clearly determined in the longitudinal and circular muscle. NK1r-IR-expressing neurons and interstitial cells were closely surrounded by substance P (SP) immunoreactive nerves. Incubation of colonic segments with nicardipine and TTX at 4 degrees C for 1 h with SP allowed to reveal a strong NK1r-IR at the surface of muscle cells. Incubation with SP (10(-6) M) at 37 degrees C for 1 min induced a relocation of NK1r-IR into the cytoplasm of muscle. This is interpreted as an internalization of NK1r induced by the binding of SP on muscular NK1r. The present data contribute to emphasize the role of NK1r in tachykinin-mediated neuronal processes regulating intestinal motility.

Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity.

PubMed

Woll, Petter S; Martin, Colin H; Miller, Jeffrey S; Kaufman, Dan S

2005-10-15

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.

Effects of butyltin exposures on MAP kinase dependent transcription regulators in human natural killer cells

PubMed Central

Person, Rachel J.; Whalen, Margaret M.

2010-01-01

Natural Killer (NK) cells are a major immune defense mechanism against cancer development and viral infection. The butyltins (BTs), tributyltin (TBT) and dibutyltin (DBT) have been widely used in industrial and other applications and significantly contaminate the environment. Both TBT and DBT have been detected in human blood. These compounds inhibit the lytic and binding function of human NK cells and thus could increase the incidence of cancer and viral infections. Butyltin (BT)-induced loss of NK function is accompanied by activation of mitogen activated protein kinases (MAPKs) and decreases in expression of cell-surface and cytolytic proteins. MAPKs activate components of the transcription regulator AP-1 and activate the transcription regulator Elk-1. Based on the fact that BTs activate MAPKs and alter protein expression, the current study examined the effect of BT exposures on the levels and phosphorylation states of the components of AP-1 and the phosphorylation state of Elk-1. Exposure to 300 nM TBT for 10 min increased the phosphorylation of c-Jun in NK cells. 1 h exposures to 300 nM and 200 nM TBT increased the phosphorylation and overall level of c-Jun. During a 300 nM treatment with TBT for 1 h the binding activity of AP-1 was significantly decreased. There were no significant alterations of AP-1 components or of Elk-1 with DBT exposures. Thus, it appears that TBT-induced alterations on phosphorylation, total levels and binding activity of c-Jun might contribute to, but are not fully responsible for, TBT-induced alterations of NK protein expression. PMID:20370538

Effects of butyltin exposures on MAP kinase-dependent transcription regulators in human natural killer cells.

PubMed

Person, Rachel J; Whalen, Margaret M

2010-06-01

Natural killer (NK) cells are a major immune defense mechanism against cancer development and viral infection. The butyltins (BTs), tributyltin (TBT) and dibutyltin (DBT), have been widely used in industrial and other applications and significantly contaminate the environment. Both TBT and DBT have been detected in human blood. These compounds inhibit the lytic and binding function of human NK cells and thus could increase the incidence of cancer and viral infections. Butyltin (BT)-induced loss of NK function is accompanied by activation of mitogen activated protein kinases (MAPKs) and decreases in expression of cell-surface and cytolytic proteins. MAPKs activate components of the transcription regulator AP-1 and activate the transcription regulator Elk-1. Based on the fact that BTs activate MAPKs and alter protein expression, the current study examined the effect of BT exposures on the levels and phosphorylation states of the components of AP-1 and the phosphorylation state of Elk-1. Exposure to 300 nM TBT for 10 min increased the phosphorylation of c-Jun in NK cells. One hour exposures to 300 nM and 200 nM TBT increased the phosphorylation and overall level of c-Jun. During a 300 nM treatment with TBT for 1 h the binding activity of AP-1 was significantly decreased. There were no significant alterations of AP-1 components or of Elk-1 with DBT exposures. Thus, it appears that TBT-induced alterations on phosphorylation, total levels, and binding activity of c-Jun might contribute to, but are not fully responsible for, TBT-induced alterations of NK protein expression.

High IFN-gamma and TNF production by peripheral NK cells of Colombian patients with different clinical presentation of Plasmodium falciparum

PubMed Central

2012-01-01

Background In Colombia, Plasmodium falciparum infection rarely results in severe disease or mortality compared to infections in African populations. During natural infection NK cells exhibit a cytolytic effect and regulate dendritic cells, macrophages, neutrophils as well as affect antigen specific T and B cell responses. To characterize the NK cells in P. falciparum infected patients of a highly endemic region of Colombia, the degree of NK proliferation and production of IFN gamma and TNF production in these cells were explored. Methods Seventeen patients with acute and three with severe P. falciparum malaria patients from the Northwest region of the country were recruited in the study. In addition, 20 healthy controls were included: 10 from Medellin (no-transmission area) and 10 from the Uraba region (a malaria endemic area). Immunophenotypic analysis of peripheral mononuclear cells was performed by FACS to detect total number of NK cells, subtypes and intracellular IFNγ and TNF production by NK cells in the different patient groups. Results The total mean CD56+/CD3- NK cell proportions in acute and severe malaria subjects were 9.14% (7.15%CD56dim, 2.01%CD56bright) and 19.62% (16.05%CD56dim, 3.58%CD56bright), respectively, in contrast to healthy controls from endemic (total mean CD56+/CD3-1.2%) and non-endemic area (total mean CD56+/CD3- 0.67%). Analysis of basal IFNγ and TNF levels confirmed the CD56bright NK population as the main cytokine producer (p < 0.0001) in the groups affected with malaria, with the CD56dim NK cell exhibiting the highest potential of TNF production after stimulus in the acute malaria group. Conclusions The results confirm the important role of not only CD56bright but also of CD56dim NK cell populations as producers of the two cytokines in malaria patients in Colombia. PMID:22316273

The proinflammatory cytokine interleukin-18 alters multiple signaling pathways to inhibit natural killer cell death

USGS Publications Warehouse

Hodge, D.L.; Subleski, J.J.; Reynolds, D.A.; Buschman, M.D.; Schill, W.B.; Burkett, M.W.; Malyguine, A.M.; Young, H.A.

2006-01-01

The proinflammatory cytokine, interleukin-18 (IL-18), is a natural killer (NK) cell activator that induces NK cell cytotoxicity and interferon-?? (IFN-??) expression. In this report, we define a novel role for IL-18 as an NK cell protective agent. Specifically, IL-18 prevents NK cell death initiated by different and distinct stress mechanisms. IL-18 reduces NK cell self-destruction during NK-targeted cell killing, and in the presence of staurosporin, a potent apoptotic inducer, IL-18 reduces caspase-3 activity. The critical regulatory step in this process is downstream of the mitochondrion and involves reduced cleavage and activation of caspase-9 and caspase-3. The ability of IL-18 to regulate cell survival is not limited to a caspase death pathway in that IL-18 augments tumor necrosis factor (TNF) signaling, resulting in increased and prolonged mRNA expression of c-apoptosis inhibitor 2 (cIAP2), a prosurvival factor and caspase-3 inhibitor, and TNF receptor-associated factor 1 (TRAF1), a prosurvival protein. The cumulative effects of IL-18 define a novel role for this cytokine as a molecular survival switch that functions to both decrease cell death through inhibition of the mitochondrial apoptotic pathway and enhance TNF induction of prosurvival factors. ?? Mary Ann Liebert, Inc.

Interaction between dendritic cells and natural killer cells during pregnancy in mice.

PubMed

Blois, Sandra M; Barrientos, Gabriela; Garcia, Mariana G; Orsal, Arif S; Tometten, Mareike; Cordo-Russo, Rosalia I; Klapp, Burghard F; Santoni, Angela; Fernández, Nelson; Terness, Peter; Arck, Petra C

2008-07-01

A complex regulation of innate and adaptive immune responses at the maternal fetal interface promotes tolerance of trophoblast cells carrying paternally derived antigens. Such regulatory functions involve uterine dendritic cells (uDC) and natural killer (uNK) cells. The existence of a NK and DC "cross talk" has been revealed in various experimental settings; its biological significance ranging from cooperative stimulation to cell lysis. Little is known about the presence or role of NK and DC cross talk at the maternal fetal interface. The present study shows that mouse NK and DC interactions are subject to modulation by trophoblast cells in vitro. This interaction promotes a tolerogenic microenvironment characterized by downregulation of the expression of activation markers on uNK cells and uDC and dominance of Th2 cytokines. NK and DC interactions would also influence uterine cell proliferation and this process would be strongly modulated by trophoblast-derived signals. Indeed; while low proliferation rates were observed upon regular coculture allowing direct contact between uterine cells and trophoblasts, incubation in a transwell culture system markedly increased uterine cell proliferation suggesting that soluble factors are key mediators in the molecular "dialog" between the mother and the conceptus during the establishment of mouse pregnancy. Our data further reveal that the regulatory functions of trophoblast cells associated with tolerance induction are impaired in high abortion murine matings. Interestingly, we observed that secretion of interleukin-12p70 by uDC is dramatically abrogated in the presence of uNK cells. Taken together, our results provide the first evidence that a delicate balance of interactions involving NK cells, DC, and trophoblasts at the mouse maternal fetal interface supports a successful pregnancy outcome.

ERAP1 regulates natural killer cell function by controlling the engagement of inhibitory receptors.

PubMed

Cifaldi, Loredana; Romania, Paolo; Falco, Michela; Lorenzi, Silvia; Meazza, Raffaella; Petrini, Stefania; Andreani, Marco; Pende, Daniela; Locatelli, Franco; Fruci, Doriana

2015-03-01

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by MHC class I (MHC-I) molecules. Herein, we demonstrate that genetic or pharmacological inhibition of ERAP1 on human tumor cell lines perturbs their ability to engage several classes of inhibitory receptors by their specific ligands, including killer cell Ig-like receptors (KIR) by classical MHC-I-peptide (pMHC-I) complexes and the lectin-like receptor CD94-NKG2A by nonclassical pMHC-I complexes, in each case leading to natural killer (NK) cell killing. The protective effect of pMHC-I complexes could be restored in ERAP1-deficient settings by the addition of known high-affinity peptides, suggesting that ERAP1 was needed to positively modify the affinity of natural ligands. Notably, ERAP1 inhibition enhanced the ability of NK cells to kill freshly established human lymphoblastoid cell lines from autologous or allogeneic sources, thereby promoting NK cytotoxic activity against target cells that would not be expected because of KIR-KIR ligand matching. Overall, our results identify ERAP1 as a modifier to leverage immune functions that may improve the efficacy of NK cell-based approaches for cancer immunotherapy. ©2015 American Association for Cancer Research.

Inhibition of bromodomain and extra-terminal (BET) proteins increases NKG2D ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in multiple myeloma cells: role of cMYC-IRF4-miR-125b interplay.

PubMed

Abruzzese, Maria Pia; Bilotta, Maria Teresa; Fionda, Cinzia; Zingoni, Alessandra; Soriani, Alessandra; Vulpis, Elisabetta; Borrelli, Cristiana; Zitti, Beatrice; Petrucci, Maria Teresa; Ricciardi, Maria Rosaria; Molfetta, Rosa; Paolini, Rossella; Santoni, Angela; Cippitelli, Marco

2016-12-01

Anti-cancer immune responses may contribute to the control of tumors after conventional chemotherapy, and different observations have indicated that chemotherapeutic agents can induce immune responses resulting in cancer cell death and immune-stimulatory side effects. Increasing experimental and clinical evidence highlight the importance of natural killer (NK) cells in immune responses toward multiple myeloma (MM), and combination therapies able to enhance the activity of NK cells against MM are showing promise in treating this hematologic cancer. The epigenetic readers of acetylated histones bromodomain and extra-terminal (BET) proteins are critical regulators of gene expression. In cancer, they can upregulate transcription of key oncogenes such as cMYC, IRF4, and BCL-2. In addition, the activity of these proteins can regulate the expression of osteoclastogenic cytokines during cancer progression. Here, we investigated the effect of BET bromodomain protein inhibition, on the expression of NK cell-activating ligands in MM cells. Five MM cell lines [SKO-007(J3), U266, RPMI-8226, ARP-1, JJN3] and CD138 + MM cells isolated from MM patients were used to investigate the activity of BET bromodomain inhibitors (BETi) (JQ1 and I-BET151) and of the selective BRD4-degrader proteolysis targeting chimera (PROTAC) (ARV-825), on the expression and function of several NK cell-activating ligands (NKG2DLs and DNAM-1Ls), using flow cytometry, real-time PCR, transient transfections, and degranulation assays. Our results indicate that inhibition of BET proteins via small molecule inhibitors or their degradation via a hetero-bifunctional PROTAC probe can enhance the expression of MICA, a ligand of the NKG2D receptor, in human MM cell lines and primary malignant plasma cells, rendering myeloma cells more efficient to activate NK cell degranulation. Noteworthy, similar results were obtained using selective CBP/EP300 bromodomain inhibition. Mechanistically, we found that BETi-mediated inhibition of cMYC correlates with the upregulation of miR-125b-5p and the downregulation of the cMYC/miR-125b-5p target gene IRF4, a transcriptional repressor of MICA. These findings provide new insights on the immuno-mediated antitumor activities of BETi and further elucidate the molecular mechanisms that regulate NK cell-activating ligand expression in MM.

Cystatin F as a regulator of immune cell cytotoxicity.

PubMed

Kos, Janko; Nanut, Milica Perišić; Prunk, Mateja; Sabotič, Jerica; Dautović, Esmeralda; Jewett, Anahid

2018-05-10

Cysteine cathepsins are lysosomal peptidases involved in the regulation of innate and adaptive immune responses. Among the diverse processes, regulation of granule-dependent cytotoxicity of cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells during cancer progression has recently gained significant attention. The function of cysteine cathepsins is regulated by endogenous cysteine protease inhibitors-cystatins. Whereas other cystatins are generally cytosolic or extracellular proteins, cystatin F is present in endosomes and lysosomes and is thus able to regulate the activity of its target directly. It is delivered to endosomal/lysosomal vesicles as an inactive, disulphide-linked dimer. Proteolytic cleavage of its N-terminal part leads to the monomer, the only form that is a potent inhibitor of cathepsins C, H and L, involved in the activation of granzymes and perforin. In NK cells and CTLs the levels of active cathepsin C and of granzyme B are dependent on the concentration of monomeric, active cystatin F. In tumour microenvironment, inactive dimeric cystatin F can be secreted from tumour cells or immune cells and further taken up by the cytotoxic cells. Subsequent monomerization and inhibition of cysteine cathepsins within the endosomal/lysosomal vesicles impairs granzyme and perforin activation, and provokes cell anergy. Further, the glycosylation pattern has been shown to be important in controlling secretion of cystatin F from target cells, as well as internalization by cytotoxic cells and trafficking to endosomal/lysosomal vesicles. Cystatin F is therefore an important mediator used by bystander cells to reduce NK and T-cell cytotoxicity.

MicroRNAs May Mediate the Down-Regulation of Neurokinin-1 Receptor in Chronic Bladder Pain Syndrome

PubMed Central

Sanchez Freire, Veronica; Burkhard, Fiona C.; Kessler, Thomas M.; Kuhn, Annette; Draeger, Annette; Monastyrskaya, Katia

2010-01-01

Bladder pain syndrome (BPS) is a clinical syndrome of pelvic pain and urinary urgency-frequency in the absence of a specific cause. Investigating the expression levels of genes involved in the regulation of epithelial permeability, bladder contractility, and inflammation, we show that neurokinin (NK)1 and NK2 tachykinin receptors were significantly down-regulated in BPS patients. Tight junction proteins zona occludens-1, junctional adherins molecule -1, and occludin were similarly down-regulated, implicating increased urothelial permeability, whereas bradykinin B1 receptor, cannabinoid receptor CB1 and muscarinic receptors M3-M5 were up-regulated. Using cell-based models, we show that prolonged exposure of NK1R to substance P caused a decrease of NK1R mRNA levels and a concomitant increase of regulatory micro(mi)RNAs miR-449b and miR-500. In the biopsies of BPS patients, the same miRNAs were significantly increased, suggesting that BPS promotes an attenuation of NK1R synthesis via activation of specific miRNAs. We confirm this hypothesis by identifying 31 differentially expressed miRNAs in BPS patients and demonstrate a direct correlation between miR-449b, miR-500, miR-328, and miR-320 and a down-regulation of NK1R mRNA and/or protein levels. Our findings further the knowledge of the molecular mechanisms of BPS, and have relevance for other clinical conditions involving the NK1 receptor. PMID:20008142

Expression of NK cell receptors on decidual T cells in human pregnancy.

PubMed

Tilburgs, Tamara; van der Mast, Barbara J; Nagtzaam, Nicole M A; Roelen, Dave L; Scherjon, Sicco A; Claas, Frans H J

2009-06-01

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.

IκBζ is essential for natural killer cell activation in response to IL-12 and IL-18

PubMed Central

Miyake, Tohru; Satoh, Takashi; Kato, Hiroki; Matsushita, Kazufumi; Kumagai, Yutaro; Vandenbon, Alexis; Tani, Tohru; Muta, Tatsushi; Akira, Shizuo; Takeuchi, Osamu

2010-01-01

IκBζ, encoded by Nfibiz, is a nuclear IκB-like protein harboring ankyrin repeats. IκBζ has been shown to regulate IL-6 production in macrophages and Th17 development in T cells. However, the role of IκBζ in natural killer (NK) cells has not be understood. In the present study, we found that the expression of IκBζ was rapidly induced in response to IL-18 in NK cells, but not in T cells. Analysis of Nfkbiz−/− mice revealed that IκBζ was essential for the production of IFN-γ production and cytotoxic activity in NK cells in response to IL-12 and/or IL-18 stimulation. IL-12/IL-18–mediated gene induction was profoundly impaired in Nfkbiz−/− NK cells. Whereas the phosphorylation of STAT4 was normally induced by IL-12 stimulation, STAT4 was not recruited to the Ifng gene regions in Nfkbiz−/− NK cells. Acetylation of histone 3 K9 on Ifng regions was also abrogated in Nfkbiz−/− NK cells. IκBζ was recruited on the proximal promoter region of the Ifng gene, and overexpression of IκBζ together with IL-12 activated the Ifng promoter. Furthermore, Nfkbiz−/− mice were highly susceptible to mouse MCMV infection. Taken together, these results demonstrate that IκBζ is essential for the activation of NK cells and antiviral host defense responses. PMID:20876105

Frontline Science: IL-18 primes murine NK cells for proliferation by promoting protein synthesis, survival, and autophagy.

PubMed

El-Darawish, Yosif; Li, Wen; Yamanishi, Kyosuke; Pencheva, Magdalena; Oka, Naoto; Yamanishi, Hiromichi; Matsuyama, Tomohiro; Tanaka, Yoshimasa; Minato, Nagahiro; Okamura, Haruki

2018-03-30

Combined stimulation by IL-2 and IL-18 effectively promotes proliferation of NK cells, whereas singular stimulation does not. In this study, synergistic effects of these cytokines on NK cells proliferation was analyzed, focusing on the roles of IL-18. In splenic resting NK cells from IL-18KO mice, IL-18 rapidly activated NF-κB independently of IL-2, and activated or up-regulated various molecules downstream of PI3K/AKT and mTOR, including S6, Bcl-XL, ATG5, and LC3II, accompanying increases in cell growth and survival. Thus, IL-18 alone was revealed to augment various cellular processes (gene transcription, protein synthesis, survival) in the absence or presence of IL-2. Notably, combined IL-18 and IL-2 promoted autophagosome formation. In addition, priming NK cells with IL-18 augmented IL-2R, especially CD25, and enabled cells to respond to IL-2, resulting in activation of STAT3 and STAT5, followed by increase of cyclin B1 leading to proliferation. However, IL-2 alone failed to activate STAT3 or STAT5 in resting IL18KO NK cells. These results clarify the distinct roles of IL-2 and IL-18 in NK cell proliferation, and the intrinsic roles of IL-18 in various cellular processes, suggesting a range of functions of IL-18 expressed in an array of nonhematopoietic cells. ©2018 Society for Leukocyte Biology.

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity.

PubMed

Tuli, Amit; Thiery, Jerome; James, Ashley M; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B; Orange, Jordan S; Lieberman, Judy; Brenner, Michael B

2013-12-01

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

Downregulation of Interleukin-18-Mediated Cell Signaling and Interferon Gamma Expression by the Hepatitis B Virus e Antigen

PubMed Central

Jegaskanda, S.; Ahn, S. H.; Skinner, N.; Thompson, A. J.; Ngyuen, T.; Holmes, J.; De Rose, R.; Navis, M.; Winnall, W. R.; Kramski, M.; Bernardi, G.; Bayliss, J.; Colledge, D.; Sozzi, V.; Visvanathan, K.; Locarnini, S. A.; Kent, S. J.

2014-01-01

ABSTRACT The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection. IMPORTANCE It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses. PMID:24872585

Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells.

PubMed

Garland, A M; Grady, E F; Payan, D G; Vigna, S R; Bunnett, N W

1994-10-01

Internalization of the NK1 receptor (NK1R) and substance P was observed in cells transfected with cDNA encoding the rat NK1R by using anti-receptor antibodies and cyanine 3-labelled substance P (cy3-substance P). After incubation at 4 degrees C, NK1R immunoreactivity and cy3-substance P were confined to the plasma membrane. Within 3 min of incubation at 37 degrees C, NK1R immunoreactivity and cy3-substance P were internalized into small intracellular vesicles located beneath the plasma membrane. Fluorescein isothiocyanate-labelled transferrin and cy3-substance P were internalized into the same vesicles, identifying them as early endosomes. After 60 min at 37 degrees C, NK1R immunoreactivity was detected in larger, perinuclear vesicles. Internalization of 125I-labelled substance P was studied by using an acid wash to dissociate cell-surface label from that which has been internalized. Binding reached equilibrium after incubation for 60 min at 4 degrees C with no detectable internalization. After 10 min incubation at 37 degrees C, 83.5 +/- 1.0% of specifically bound counts were internalized. Hyperosmolar sucrose and phenylarsine oxide, which are inhibitors of endocytosis, prevented internalization of 125I-labelled substance P and accumulation of NK1R immunoreactivity into endosomes. Acidotropic agents caused retention of 125I-labelled substance P within the cell and inhibited degradation of the internalized peptide. Continuous incubation of cells with substance P at 37 degrees C reduced 125I-substance P binding at the cell surface. Therefore, substance P and its receptor are internalized into early endosomes within minutes of binding, and internalized substance P is degraded. Internalization depletes NK1Rs from the cell surface and may down-regulate the response of a cell to substance P.

The PD-1/B7-H1 pathway modulates the natural killer cells versus mouse glioma stem cells.

PubMed

Huang, Bo Yuan; Zhan, Yi Ping; Zong, Wen Jing; Yu, Chun Jiang; Li, Jun Fa; Qu, Yan Ming; Han, Song

2015-01-01

Glioblastoma multiforme (GBM) is the most malignant primary type of brain tumor in adults. There has been increased focus on the immunotherapies to treat GBM patients, the therapeutic value of natural killer (NK) cells is still unknown. Programmed death-1 (PD-1) is a major immunological checkpoint that can negatively regulate the T-cell-mediated immune response. We tested the combination of the inhibiting the PD-1/B7H1 pathway with a NK-cell mediated immune response in an orthotopic mouse model of GBM. Mouse glioma stem cells (GL261GSCs) and mouse NK cells were isolated and identified. A lactate dehydrogenase (LDH) assay was perfomed to detect the cytotoxicity of NK cells against GL261GSCs. GL261GSCs were intracranially implanted into mice, and the mice were stratified into 3 treatment groups: 1) control, 2) NK cells treatment, and 3) PD-1 inhibited NK cells treatment group. Overall survival was quantified, and animal magnetic resonance imaging (MRI) was performed to determine tumor growth. The brains were harvested after the mice were euthanized, and immunohistochemistry against CD45 and PCNA was performed. The mouse NK cells were identified as 90% CD3- NK1.1+CD335+ by flow cytometric analysis. In the LDH assay, the ratios of the damaged GL261GSCs, with the E:T ratios of 2.5:1, 5:1, and 10:1, were as follows: 1) non-inhibited group: 7.42%, 11.31%, and 15.1%, 2) B7H1 inhibited group: 14.75%, 18.25% and 29.1%, 3) PD-1 inhibited group: 15.53%, 19.21% and 29.93%, 4) double inhibited group: 33.24%, 42.86% and 54.91%. In the in vivo experiments, the mice in the PD-1 inhibited NK cells treatment group and IL-2-stimulated-NK cells treatment group displayed a slowest tumor growth (F = 308.5, P<0.01) and a slower tumor growth compared with control group (F = 118.9, P<0.01), respectively. The median survival of the mice in the three groups were as follows: 1) conrol group: 29 days, 2) NK cells treatment group: 35 days (P = 0.0012), 3) PD-1 inhibited NK cells treatment group: 44 days (P = 0.0024). Immunologic data of PCNA-positive cell ratios and CD45-positive cell ratios of the tumor specimens in the three groups were as follows: 1) control group: 65.72% (PCNA) and 0.92% (CD45), 2) NK treatment group: 27.66% (PCNA) and 13.46% (CD45), and 3) PD-1 inhibited NK cells treatment group: 13.66% (PCNA) and 23.66% (CD45) (P<0.001). The results demonstrated that blockade of PD-1/B7H1 pathway could promote mouse NK cells to kill the GL261GSCs, and the PD-1-inhibited NK cells could be a feasible immune therapeutic approach against GBM.

Unusual Suspects in the Development of Obesity-Induced Inflammation and Insulin Resistance: NK cells, iNKT cells, and ILCs.

PubMed

Bonamichi, Beatriz Dal Santo Francisco; Lee, Jongsoon

2017-08-01

The notion that obesity-induced inflammation mediates the development of insulin resistance in animal models and humans has been gaining strong support. It has also been shown that immune cells in local tissues, in particular in visceral adipose tissue, play a major role in the regulation of obesity-induced inflammation. Specifically, obesity increases the numbers and activation of proinflammatory immune cells, including M1 macrophages, neutrophils, Th1 CD4 T cells, and CD8 T cells, while simultaneously suppressing anti-inflammatory cells such as M2 macrophages, CD4 regulatory T cells, regulatory B cells, and eosinophils. Recently, however, new cell types have been shown to participate in the development of obesity-induced inflammation and insulin resistance. Some of these cell types also appear to regulate obesity. These cells are natural killer (NK) cells and innate lymphoid cells (ILCs), which are closely related, and invariant natural killer T (iNKT) cells. It should be noted that, although iNKT cells resemble NK cells in name, they are actually a completely different cell type in terms of their development and functions in immunity and metabolism. In this review, we will focus on the roles that these relatively new players in the metabolism field play in obesity-induced insulin resistance and the regulation of obesity. Copyright © 2017 Korean Diabetes Association.

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

Anticancer drugs cause release of exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective natural killer cell antitumor responses in vitro.

PubMed

Lv, Li-Hong; Wan, Yun-Le; Lin, Yan; Zhang, Wei; Yang, Mei; Li, Guo-Lin; Lin, Hao-Ming; Shang, Chang-Zhen; Chen, Ya-Jin; Min, Jun

2012-05-04

Failure of immune surveillance related to inadequate host antitumor immune responses has been suggested as a possible cause of the high incidence of recurrence and poor overall survival outcome of hepatocellular carcinoma. The stress-induced heat shock proteins (HSPs) are known to act as endogenous "danger signals" that can improve tumor immunogenicity and induce natural killer (NK) cell responses. Exosome is a novel secretory pathway for HSPs. In our experiments, the immune regulatory effect of the HSP-bearing exosomes secreted by human hepatocellular carcinoma cells under stress conditions on NK cells was studied. ELISA results showed that the production of HSP60, HSP70, and HSP90 was up-regulated in both cell lines in a stress-specific manner. After exposure to hepatocellular carcinoma cell-resistant or sensitive anticancer drugs (hereafter referred to as "resistant" or "sensitive" anticancer drug), the membrane microvesicles were actively released by hepatocellular carcinoma cells, differing in their ability to present HSPs on the cell surface, which were characterized as exosomes. Acting as a decoy, the HSP-bearing exosomes efficiently stimulated NK cell cytotoxicity and granzyme B production, up-regulated the expression of inhibitory receptor CD94, and down-regulated the expression of activating receptors CD69, NKG2D, and NKp44. Notably, resistant anticancer drugs enhanced exosome release and generated more exosome-carried HSPs, which augmented the activation of the cytotoxic response. In summary, our findings demonstrated that exosomes derived from resistant anticancer drug-treated HepG2 cells conferred superior immunogenicity in inducing HSP-specific NK cell responses, which provided a clue for finding an efficient vaccine for hepatocellular carcinoma immunotherapy.

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell–mediated cytotoxicity

PubMed Central

Tuli, Amit; Thiery, Jerome; James, Ashley M.; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B.; Orange, Jordan S.; Lieberman, Judy; Brenner, Michael B.

2013-01-01

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell–mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity. PMID:24088571

Constitutively polarized granules prime KHYG-1 NK cells.

PubMed

Suck, Garnet; Branch, Donald R; Aravena, Paola; Mathieson, Mark; Helke, Simone; Keating, Armand

2006-09-01

The major mechanism for NK cell lysis of tumor cells is granule-mediated cytotoxicity. Polarization of granules is a prelude to the release of their cytotoxic contents in response to target-cell binding. We describe the novel observation of constitutive granule polarization in the cytotoxic NK cell line, KHYG-1. Continuous degranulation of KHYG-1 cells, however, does not occur and still requires target-cell contact. Disruption of microtubules with colcemid is sufficient to disperse the granules in KHYG-1 and significantly decreases cytotoxicity. A similar effect is not obtained by inhibiting extracellular signal-related kinase 2 (ERK2), the most distal kinase investigated in the cytolytic pathway. Disruption of microtubules significantly down-regulates activation receptors, NKp44 and NKG2D, implicating them as potential microtubule-trafficking receptors. Such changes in upstream receptor expression may have caused deactivation of ERK2, since NKG2D cross-linking also leads to receptor down-regulation and diminished ERK phosphorylation. Thus, a functional role for NKG2D in KHYG-1 cytotoxicity is demonstrated. Moreover, the novel primed state may contribute to the high cytotoxicity exhibited by KHYG-1.

Mesenchymal Stromal Cells Prevent Allostimulation In Vivo and Control Checkpoints of Th1 Priming: Migration of Human DC to Lymph Nodes and NK Cell Activation.

PubMed

Consentius, C; Akyüz, L; Schmidt-Lucke, J A; Tschöpe, C; Pinzur, L; Ofir, R; Reinke, P; Volk, H-D; Juelke, K

2015-10-01

Although the immunomodulatory potency of mesenchymal stromal cells (MSC) is well established, the mechanisms behind are still not clear. The crosstalk between myeloid dendritic cells (mDC) and natural killer (NK) cells and especially NK cell-derived interferon-gamma (IFN-γ) play a pivotal role in the development of type 1 helper (Th1) cell immune responses. While many studies explored the isolated impact of MSC on either in vitro generated DC, NK, or T cells, there are only few data available on the complex interplay between these cells. Here, we investigated the impact of MSC on the functionality of human mDC and the consequences for NK cell and Th1 priming in vitro and in vivo. In critical limb ischemia patients, who have been treated with allogeneic placenta-derived mesenchymal-like stromal cells (PLX-PAD), no in vivo priming of Th1 responses toward the major histocompatibility complex (MHC) mismatches could be detected. Further in vitro studies revealed that mDC reprogramming could play a central role for these effects. Following crosstalk with MSC, activated mDC acquired a tolerogenic phenotype characterized by reduced migration toward CCR7 ligand and impaired ability to stimulate NK cell-derived IFN-γ production. These effects, which were strongly related to an altered interleukin (IL)-12/IL-10 production by mDC, were accompanied by an effective prevention of Th1 priming in vivo. Our findings provide novel evidence for the regulation of Th1 priming by MSC via modulation of mDC and NK cell crosstalk and show that off-the-shelf produced MHC-mismatched PLX-PAD can be used in patients without any sign of immunogenicity. © 2015 AlphaMed Press.

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

Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells

PubMed Central

Sojka, Dorothy K; Plougastel-Douglas, Beatrice; Yang, Liping; Pak-Wittel, Melissa A; Artyomov, Maxim N; Ivanova, Yulia; Zhong, Chao; Chase, Julie M; Rothman, Paul B; Yu, Jenny; Riley, Joan K; Zhu, Jinfang; Tian, Zhigang; Yokoyama, Wayne M

2014-01-01

Natural killer (NK) cells belong to the innate immune system; they can control virus infections and developing tumors by cytotoxicity and producing inflammatory cytokines. Most studies of mouse NK cells, however, have focused on conventional NK (cNK) cells in the spleen. Recently, we described two populations of liver NK cells, tissue-resident NK (trNK) cells and those resembling splenic cNK cells. However, their lineage relationship was unclear; trNK cells could be developing cNK cells, related to thymic NK cells, or a lineage distinct from both cNK and thymic NK cells. Herein we used detailed transcriptomic, flow cytometric, and functional analysis and transcription factor-deficient mice to determine that liver trNK cells form a distinct lineage from cNK and thymic NK cells. Taken together with analysis of trNK cells in other tissues, there are at least four distinct lineages of NK cells: cNK, thymic, liver (and skin) trNK, and uterine trNK cells. DOI: http://dx.doi.org/10.7554/eLife.01659.001 PMID:24714492

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

Saïdi, Héla; Bras, Marlène; Formaglio, Pauline; Melki, Marie-Thérèse; Charbit, Bruno; Herbeuval, Jean-Philippe; Gougeon, Marie-Lise

2016-02-01

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 TRAIL expression at the surface of pDCs and NK cells, and they suggest a novel mechanism of innate control of HIV-1 infection.

Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor

PubMed Central

1994-01-01

Interleukin 15 (IL-15) is a novel cytokine that has recently been cloned and expressed. Whereas it has no sequence homology with IL-2, IL- 15 interacts with components of the IL-2 receptor (IL-2R). In the present study we performed a functional analysis of recombinant IL-15 on phenotypically and functionally distinct populations of highly purified human natural killer (NK) cells. The CD56bright subset of human NK cells constitutively expresses the high affinity IL-2R and exhibits a brisk proliferative response after the binding of picomolar amounts of IL-2. Using a proliferation assay, IL-15 demonstrated a very steep dose-response curve that was distinct from the dose-response curve for IL-2. The proliferative effects of IL-15 could be abrogated by anti-IL-2R beta (p75), but not by anti-IL-2R alpha (p55). The proliferative effects of IL-2 on CD56bright NK cells could be inhibited by both antibodies. CD56dim NK cells express the intermediate affinity IL-2R in the absence of the high affinity IL-2R. Activation of CD56dim NK cells by IL-15 was similar to that of IL-2 as measured by enhanced NK cytotoxic activity, antibody-dependent cellular cytotoxicity, and NK cell production of interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor. The IL-15-enhanced NK cytotoxic activity could be completely blocked by anti-IL-2R beta monoclonal antibody. The binding of radiolabeled IL-2 and IL-15 to CD56dim NK cells was inhibited in the presence of anti-IL-2R beta. Scatchard analysis of radiolabeled IL-15 and IL-2 binding to NK- enriched human lymphocytes revealed the presence of high and intermediate affinity receptors for both ligands. IL-15 is a ligand that activates human NK cells through components of the IL-2R in a pattern that is similar but not identical to that of IL-2. Unlike IL-2, IL-15 is produced by activated monocytes/macrophages. The discovery of IL-15 may increase our understanding of how monocytes/macrophages participate in the regulation of NK cell function. PMID:7523571

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 TRAIL expression at the surface of pDCs and NK cells, and they suggest a novel mechanism of innate control of HIV-1 infection. PMID:26871575

Sub-apoptotic dosages of pro-oxidant vitamin cocktails sensitize human melanoma cells to NK cell lysis.

PubMed

Tremante, Elisa; Santarelli, Lory; Lo Monaco, Elisa; Sampaoli, Camilla; Ingegnere, Tiziano; Guerrieri, Roberto; Tomasetti, Marco; Giacomini, Patrizio

2015-10-13

Alpha-tocopheryl succinate (αTOS), vitamin K3 (VK3) and vitamin C (ascorbic acid, AA) were previously shown to synergistically promote different death pathways in carcinoma cells, depending on their concentrations and combinations. Similar effects were observed herein in melanoma cells, although αTOS behaved as an antagonist. Interestingly, suboptimal cell death-inducing concentrations (1.5 μM αTOS/20 μM AA/0.2 μM VK3) effectively up-regulated activating Natural Killer (NK) cell ligands, including MICA (the stress-signaling ligand of the NKG2D receptor), and/or the ligands of at least one of the natural cytotoxicity receptors (NKp30, NKp44 and NKp46) in 5/6 melanoma cell lines. Only an isolated MICA down-regulation was seen. HLA class I, HLA class II, ULBP1, ULBP2, ULBP3, Nectin-2, and PVR displayed little, if any, change in expression. Ligand up-regulation resulted in improved lysis by polyclonal NK cells armed with the corresponding activating receptors. These results provide the first evidence for concerted induction of cell death by cell-autonomous and extrinsic (immune) mechanisms. Alarming the immune system much below the cell damage threshold may have evolved as a sensitive readout of neoplastic transformation and oxidative stress. Cocktails of vitamin analogues at slightly supra-physiological dosages may find application as mild complements of melanoma treatment, and in chemoprevention.

Sub-apoptotic dosages of pro-oxidant vitamin cocktails sensitize human melanoma cells to NK cell lysis

PubMed Central

Tremante, Elisa; Santarelli, Lory; Monaco, Elisa Lo; Sampaoli, Camilla; Ingegnere, Tiziano; Guerrieri, Roberto

2015-01-01

Alpha-tochopheryl succinate (αTOS), vitamin K3 (VK3) and vitamin C (ascorbic acid, AA) were previously shown to synergistically promote different death pathways in carcinoma cells, depending on their concentrations and combinations. Similar effects were observed herein in melanoma cells, although αTOS behaved as an antagonist. Interestingly, suboptimal cell death-inducing concentrations (1.5 μM αTOS/20 μM AA/0.2 μM VK3) effectively up-regulated activating Natural Killer (NK) cell ligands, including MICA (the stress-signaling ligand of the NKG2D receptor), and/or the ligands of at least one of the natural cytotoxicity receptors (NKp30, NKp44 and NKp46) in 5/6 melanoma cell lines. Only an isolated MICA down-regulation was seen. HLA class I, HLA class II, ULBP1, ULBP2, ULBP3, Nectin-2, and PVR displayed little, if any, change in expression. Ligand up-regulation resulted in improved lysis by polyclonal NK cells armed with the corresponding activating receptors. These results provide the first evidence for concerted induction of cell death by cell-autonomous and extrinsic (immune) mechanisms. Alarming the immune system much below the cell damage threshold may have evolved as a sensitive readout of neoplastic transformation and oxidative stress. Cocktails of vitamin analogues at slightly supra-physiological dosages may find application as mild complements of melanoma treatment, and in chemoprevention. PMID:26427039

KIR and HLA-C Interactions Promote Differential Dendritic Cell Maturation and Is a Major Determinant of Graft Failure following Kidney Transplantation

PubMed Central

Hanvesakul, Raj; Kubal, Chandrashekhar; Moore, Jason; Neil, Desley; Cook, Mark; Ball, Simon; Briggs, David; Moss, Paul; Cockwell, Paul

2011-01-01

Background HLA-C is an important ligand for killer immunoglobulin like receptors (KIR) that regulate natural killer (NK) cell function. Based on KIR specificity HLA-C molecules are allocated into two groups, HLA-C1 or HLA-C2; HLA-C2 is more inhibiting to NK cell function than HLA-C1. We studied the clinical importance of HLA-C genotypes on the long-term graft survival of 760 kidney transplants performed at our centre utilising a population based genetic study and cell culture model to define putative mechanisms. Methods and Findings Genotyping was performed using conventional DNA PCR techniques and correlations made to clinical outcomes. We found that transplant recipients with HLA-C2 had significantly better long-term graft survival than transplant recipients with HLA-C1 (66% versus 44% at 10 years, log-rank p = 0.002, HR = 1.51, 95%CI = 1.16–1.97). In in-vitro NK and dendritic cell (DC) co-culture model we made several key observations that correlated with the population based genetic study. We observed that donor derived NK cells, on activation with IL-15, promoted differential HLA-C genotype dependent DC maturation. In NK-DC co-culture, the possession of HLA-C2 by DC was associated with anti-inflammatory cytokine production (IL-1RA/IL-6), diminished DC maturation (CD86, HLA-DR), and absent CCR7 expression. Conversely, possession of HLA-C1 by DC was associated with pro-inflammatory cytokine synthesis (TNF-α, IL-12p40/p70), enhanced DC maturation and up-regulation of CCR7 expression. By immunohistochemistry the presence of donor NK cells was confirmed in pre-transplant kidneys. Conclusions We propose that after kidney transplantation IL-15 activated donor derived NK cells interact with recipient DC with less activation of indirect allo-reactivity in HLA-C2 positive recipients than HLA-C1 positive recipients; this has implications for long-term graft survival. Early events following kidney transplantation involving NK-DC interaction via KIR and HLA-C immune synapse may have a central role in long-term kidney transplant outcomes. PMID:21912600

Inhibition of histone H3K27 demethylases selectively modulates inflammatory phenotypes of natural killer cells.

PubMed

Cribbs, Adam; Hookway, Edward S; Wells, Graham; Lindow, Morten; Obad, Susanna; Oerum, Henrik; Prinjha, Rab K; Athanasou, Nick; Sowman, Aneka; Philpott, Martin; Penn, Henry; Soderstrom, Kalle; Feldmann, Marc; Oppermann, Udo

2018-02-16

Natural killer (NK) cells are innate lymphocytes, important in immune surveillance and elimination of stressed, transformed, or virus-infected cells. They critically shape the inflammatory cytokine environment to orchestrate interactions of cells of the innate and adaptive immune systems. Some studies have reported that NK cell activation and cytokine secretion are controlled epigenetically but have yielded only limited insight into the mechanisms. Using chemical screening with small-molecule inhibitors of chromatin methylation and acetylation, further validated by knockdown approaches, we here identified Jumonji-type histone H3K27 demethylases as key regulators of cytokine production in human NK cell subsets. The prototypic JMJD3/UTX (Jumonji domain-containing protein 3) H3K27 demethylase inhibitor GSK-J4 increased global levels of the repressive H3K27me3 mark around transcription start sites of effector cytokine genes. Moreover, GSK-J4 reduced IFN-γ, TNFα, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-10 levels in cytokine-stimulated NK cells while sparing their cytotoxic killing activity against cancer cells. The anti-inflammatory effect of GSK-J4 in NK cell subsets, isolated from peripheral blood or tissue from individuals with rheumatoid arthritis (RA), coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggested that histone demethylase inhibition has broad utility for modulating immune and inflammatory responses. Overall, our results indicate that H3K27me3 is a dynamic and important epigenetic modification during NK cell activation and that JMJD3/UTX-driven H3K27 demethylation is critical for NK cell function. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

[Immune regulation activity and mechanism of Tibetan Kefir exopolysaccharide fractions].

PubMed

Meng, Li; Zhang, Lanwei

2009-12-01

To investigate the effects and mechanism on immune regulation activity in mice of two Tibetan Kefir exoploysaccharides (EPS) with different molecular weight of 0.1 x 10(5) - 3 x 10(5) (fraction 1) and 1.8 x 10(3) (fraction 2). The immune regulation activity experiment was carried out in vitro based on the Functional Assessment Procedure and Test Methods of Health Food, which was issued by Ministry of Health of China. First, we treated mice subjects with EPS at doses of 40 mg/kg, 80 mg/kg, 120 mg/kg through ig. Then we detected the index of immune organs, the ability of antibody production (tested by HC50), activity of NK cell, delayed type hypersensitivity (DTH) and phagocytosis of macrophage in mice. Finally, we examined the expression of Erk protein in Macrophages by Western Blot assay. Fraction 1 could promote HC50, activity of NK cell and DTH in mice which low dose showed better. Fraction 2 could promote DTH, phagocytosis of macrophage which high dose showed better. The expression of Erk and COX-2 had the same trend with Phagocytic index. We verified the two fractions of Tibetan Kefir EPS could enhance immune functions in mice. Fraction 1 regulated immune function through NK cell and B cell while fraction 2 through macrophage cell and T cell. The effects to macrophage of Tibetan Kefir EPS in mice may realize through extra cellular signal-regulated kinase Erk pathway.

Interplay between Natural Killer Cells and Anti-HER2 Antibodies: Perspectives for Breast Cancer Immunotherapy

PubMed Central

Muntasell, Aura; Cabo, Mariona; Servitja, Sonia; Tusquets, Ignasi; Martínez-García, María; Rovira, Ana; Rojo, Federico; Albanell, Joan; López-Botet, Miguel

2017-01-01

Overexpression of the human epidermal growth factor receptor 2 (HER2) defines a subgroup of breast tumors with aggressive behavior. The addition of HER2-targeted antibodies (i.e., trastuzumab, pertuzumab) to chemotherapy significantly improves relapse-free and overall survival in patients with early-stage and advanced disease. Nonetheless, considerable proportions of patients develop resistance to treatment, highlighting the need for additional and co-adjuvant therapeutic strategies. HER2-specific antibodies can trigger natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity and indirectly enhance the development of tumor-specific T cell immunity; both mechanisms contributing to their antitumor efficacy in preclinical models. Antibody-dependent NK cell activation results in the release of cytotoxic granules as well as the secretion of pro-inflammatory cytokines (i.e., IFNγ and TNFα) and chemokines. Hence, NK cell tumor suppressive functions include direct cytolytic killing of tumor cells as well as the regulation of subsequent antitumor adaptive immunity. Albeit tumors with gene expression signatures associated to the presence of cytotoxic lymphocyte infiltrates benefit from trastuzumab-based treatment, NK cell-related biomarkers of response/resistance to HER2-specific therapeutic antibodies in breast cancer patients remain elusive. Several variables, including (i) the configuration of the patient NK cell repertoire; (ii) tumor molecular features (i.e., estrogen receptor expression); (iii) concomitant therapeutic regimens (i.e., chemotherapeutic agents, tyrosine kinase inhibitors); and (iv) evasion mechanisms developed by progressive breast tumors, have been shown to quantitatively and qualitatively influence antibody-triggered NK cell responses. In this review, we discuss possible interventions for restoring/enhancing the therapeutic activity of HER2 therapeutic antibodies by harnessing NK cell antitumor potential through combinatorial approaches, including immune checkpoint blocking/stimulatory antibodies, cytokines and toll-like receptor agonists. PMID:29181007

Expansions of NK-like αβT cells with chronologic aging: Novel lymphocyte effectors that compensate for functional deficits of conventional NK cells and T cells

PubMed Central

Vallejo, Abbe N.; Mueller, Robert G.; Hamel, David L.; Way, Amanda; Dvergsten, Jeffrey A.; Griffin, Patricia; Newman, Anne B.

2010-01-01

As the repertoire of αβT cell receptors (TCR) contracts with advancing age, there is an associated age-dependent accumulation of oligoclonal T cells expressing of a variety of receptors (NKR), normally expressed on natural killer (NK) cells. Evidences for differential regulation of expression of particular NKRs between T cells and NK cells suggest that NKR expression on T cells is physiologically programmed rather than a random event of the aging process. Experimental studies show NKRs on aged αβT cells may function either as independent receptors, and/or as costimulatory receptors to the TCR. Considering the reported deficits of conventional αβTCR-driven activation and also functional deficits of classical NK cells, NKR+ αβT cells likely represent novel immune effectors that are capable of combining innate and adaptive functions. Inasmuch as immunity is a determinant of individual fitness, the type and density of NKRs could be important contributing factors to the wide heterogeneity of health characteristics of older adults, ranging from institutionalized frail elders who are unable to mount immune responses to functionally independent community-dwelling elders who exhibit protective immunity. Understanding the biology of NKR+ αβT cells could lead to new avenues for age-specific intervention to improve protective immunity. PMID:20932941

Neuropeptides activate human mast cell degranulation and chemokine production

PubMed Central

Kulka, Marianna; Sheen, Cecilia H; Tancowny, Brian P; Grammer, Leslie C; Schleimer, Robert P

2008-01-01

During neuronal-induced inflammation, mast cells may respond to stimuli such as neuropeptides in an FcεRI-independent manner. In this study, we characterized human mast cell responses to substance P (SP), nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and compared these responses to human mast cell responses to immunoglobulin E (IgE)/anti-IgE and compound 48/80. Primary cultured mast cells, generated from CD34+ progenitors in the presence of stem cell factor and interleukin-6 (IL-6), and human cultured mast cells (LAD2) were stimulated with these and other stimuli (gastrin, concanavalin A, radiocontrast media, and mannitol) and their degranulation and chemokine production was assessed. VIP and SP stimulated primary human mast cells and LAD cells to degranulate; gastrin, concanavalin A, radiocontrast media, mannitol, CGRP and NGF did not activate degranulation. While anti-IgE stimulation did not induce significant production of chemokines, stimulation with VIP, SP or compound 48/80 potently induced production of monocyte chemoattractant protein-1, inducible protein-10, monokine induced by interferon-γ (MIG), RANTES (regulated on activation, normal, T-cell expressed, and secreted) and IL-8. VIP, SP and compound 48/80 also activated release of tumour necrosis factor, IL-3 and granulocyte–macrophage colony-stimulating factor, but not IL-4, interferon-γ or eotaxin. Human mast cells expressed surface neurokinin 1 receptor (NK1R), NK2R, NK3R and VIP receptor type 2 (VPAC2) but not VPAC1 and activation of human mast cells by IgE/anti-IgE up-regulated expression of VPAC2, NK2R, and NK3R. These studies demonstrate the pattern of receptor expression and activation of mast cell by a host of G-protein coupled receptor ligands and suggest that SP and VIP activate a unique signalling pathway in human mast cells. These results are likely to have direct relevance to neuronally induced inflammatory diseases. PMID:17922833

Estrogen upregulates MICA/B expression in human non-small cell lung cancer through the regulation of ADAM17.

PubMed

Ren, Jing; Nie, Yunzhong; Lv, Mingming; Shen, Sunan; Tang, Ruijing; Xu, Yujun; Hou, Yayi; Zhao, Shuli; Wang, Tingting

2015-11-01

Estrogen is involved in promoting lung cancer cell division and metastasis. MICA and MICB function as ligands for NKG2D, an important immunoreceptor expressed on natural killer (NK) cells. However, whether estrogen regulates MICA/B expression and affects tumor immune escape remains unknown. In this study, we measured the mRNA levels of MICA, MICB and ADAM17in non-small cell lung cancer (NSCLC) cell lines treated with estrogen. Surface expression of MICA/B on LTEP-a2 and A549 was detected using flow cytometry. We demonstrate that both mRNA and secretory protein levels of MICA/B in lung adenocarcinoma cell lines were upregulated by estradiol. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. This secretion of MICA/B downregulated the NKG2D receptor on the surface of NK92 cells and impaired the cytotoxic activity of NK cells. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. Furthermore, a significant correlation between the concentration of estradiol and the expression of MICA was found in tumor tissues of NSCLC patients. Therefore, we conclude that estrogen can regulate the expression and secretion of MICA/B through ADAM17, which helps lung cancer cells escape NKG2D-mediated immune surveillance.

NK1R/5-HT1AR interaction is related to the regulation of melanogenesis.

PubMed

Wu, Huali; Zhao, Yucheng; Huang, Qiaoling; Cai, Minxuan; Pan, Qi; Fu, Mengsi; An, Xiaohong; Xia, Zhenjiang; Liu, Meng; Jin, Yu; He, Ling; Shang, Jing

2018-06-01

Substance P (SP) is a candidate mediator along the brain-skin axis and can mimic the effects of stress to regulate melanogenesis. Previously, we and others have found that the regulation of SP for pigmentary function was mediated by neurokinin 1 receptor (NK1R). Emerging evidence has accumulated that psychologic stress can induce dysfunction in the cutaneous serotonin 5-hydroxytryptamine (5-HT)-5-HT1A/1B receptor system, thereby resulting in skin hypopigmentation. Moreover, NK1R and 5-HTR (except 5-HT3) belong to GPCR. The present study aimed at assessing the possible existence of NK1R-5-HTR interactions and related melanogenic functions. Western blot and PCR detection revealed that SP reduced expression of 5-HT1A receptor via the NK1 receptor. Biochemical analyses showed that NK1R and 5-HT1AR could colocalize and interact in a cell and in the skin. When the N terminus of the NK1R protein was removed NK1R surface targeting was prevented, the interaction between NK1R-5-HT1AR decreased, and the depigmentation caused by SP and WAY100635 could be rescued. Importantly, pharmaceutical coadministration of NK1R agonist (SP) and 5-HT1A antagonist (WAY100635) enhanced the NK1-5-HT1A receptor coimmunoprecipitation along with the depigmentary response. SP and WAY100635 cooperation elicited activation of a signaling cascade (the extracellular, regulated protein kinase p-JNK signaling pathway) and inhibition of p70S6K1 phosphorylation and greatly reduced melanin production in vitro and in vivo in mice and zebrafish. Moreover, the SP-induced depigmentation response did not be occur in 5-htr1aa +/- zebrafish embryos. Taken together, the results of our systemic study increases our knowledge of the roles of NK1R and 5-HT1AR in melanogenesis and provides possible, novel therapeutic strategies for treatment of skin hypo/hyperpigmentation.-Wu, H., Zhao, Y., Huang, Q., Cai, M., Pan, Q., Fu, M., An, X., Xia, Z., Liu, M., Jin, Y., He, L., Shang, J. NK1R/5-HT1AR interaction is related to the regulation of melanogenesis.

Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcet-Palacios, Marcelo; Odemuyiwa, Solomon O.; Coughlin, Jason J.

2008-02-15

Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Ourmore » data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 {mu}g/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.« less

Structure-guided design of an invariant natural killer T cell agonist for optimum protection from type 1 diabetes in non-obese diabetic mice

PubMed Central

Blumenfeld, H J; Tohn, R; Haeryfar, S M M; Liu, Y; Savage, P B; Delovitch, T L

2011-01-01

Because invariant natural killer T (iNK T) cells link innate and adaptive immunity, the structure-dependent design of iNK T cell agonists may have therapeutic value as vaccines for many indications, including autoimmune disease. Previously, we showed that treatment of non-obese diabetic (NOD) mice with the iNK T cell activating prototypic glycolipid α-galactosylceramide (α-GalCer) protects them from type 1 diabetes (T1D). However, α-GalCer is a strong agonist that can hyperactivate iNK T cells, elicit several side effects and has shown only limited success in clinical trials. Here, we used a structure-guided design approach to identify an iNK T cell agonist that optimally protects from T1D with minimal side effects. Analyses of the kinetics and function of a panel of synthetic α-GalCer fatty acyl chain derivatives (C8:0-C16:0) were performed in NOD mice. C16:0 elicited the highest protection from insulitis and T1D, which was associated with a higher frequency and survival of iNK T cells and enhanced activity of tolerogenic dendritic cells (DCs) in draining pancreatic lymph nodes (PLN), inability to transactivate NK cells and a more rapid kinetics of induction and recovery of iNK T cells from anergy. We conclude that the length and structure of the acyl chain of α-GalCer regulates the level of protection against T1D in mice, and propose that the extent of this protection depends on the relative capacity of the acyl chain to accommodate an endogenous spacer lipid of appropriate length and structure. Thus, our findings with the α-GalCer C16:0 derivative suggest strongly that it be considered as a lead glycolipid candidate in clinical trials of T1D. PMID:21910729

A synergistic interferon-γ production is induced by mouse hepatitis virus in interleukin-12 (IL-12)/IL-18-activated natural killer cells and modulated by carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 1a receptor

PubMed Central

Jacques, Alexandre; Bleau, Christian; Turbide, Claire; Beauchemin, Nicole; Lamontagne, Lucie

2009-01-01

The production of interferon-γ (IFN-γ) by infiltrating natural killer (NK) cells in liver is involved in the control of mouse hepatitis virus (MHV) infection. The objectives of this study were to identify the mechanisms used by MHV type 3 to modulate the production of IFN-γ by NK cells during the acute hepatitis in susceptible C57BL/6 mice. Ex vivo and in vitro experiments revealed that NK cells, expressing carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 1a (the MHV receptor), can produce a higher level of IFN-γ in the presence of both L2-MHV3 and interleukin-12 (IL-12)/IL-18. The synergistic production of IFN-γ by NK cells depends on viral replication rather than viral fixation only, because it is inhibited or not induced in cells infected with ultraviolet-inactivated viruses and in cells from Ceacam1a−/− mice infected with virulent viruses. The synergistic IFN-γ production involves the p38 mitogen-activated protein kinase (MAPK) rather than the extracellular signal-regulated kinase-1/2 MAPK signalling pathway. However, the signal triggered through the engagement of CEACAM1a decreases the production of IFN-γ, when these molecules are cross-linked using specific monoclonal antibodies. These results suggest that control of acute hepatitis by IFN-γ-producing NK cells may depend on both production of IL-12 and IL-18 in the liver environment and viral infection of NK cells. PMID:19740316

Adhesion of Epstein–Barr virus-positive natural killer cell lines to cultured endothelial cells stimulated with inflammatory cytokines

PubMed Central

Kanno, H; Watabe, D; Shimizu, N; Sawai, T

2008-01-01

Chronic active Epstein–Barr virus (EBV) infection (CAEBV) is characterized by chronic recurrent infectious mononucleosis-like symptoms. Approximately one-fourth of CAEBV patients develop vascular lesions with infiltration of EBV-positive lymphoid cells. Furthermore, EBV-positive natural killer (NK)/T cell lymphomas often exhibit angiocentric or angiodestructive lesions. These suggest an affinity of EBV-positive NK/T cells to vascular components. In this study, we evaluated the expression of adhesion molecules and cytokines in EBV-positive NK lymphoma cell lines, SNK1 and SNK6, and examined the role of cytokines in the interaction between NK cell lines and endothelial cells. SNKs expressed intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) at much higher levels than those in EBV-negative T cell lines. SNKs produced the larger amount of tumour necrosis factor (TNF)-α, which caused increased expression of ICAM-1 and VCAM-1 in cultured human endothelial cells, than that from EBV-negative T cell lines. Furthermore, SNKs exhibited increased adhesion to cultured endothelial cells stimulated with TNF-α or interleukin (IL)-1β, and the pretreatment of cytokine-stimulated endothelial cells with anti-VCAM-1-antibodies reduced cell adhesion. These indicate that the up-regulated expression of VCAM-1 on cytokine-stimulated endothelial cells would be important for the adhesion of EBV-positive NK cells and might initiate the vascular lesions. PMID:18190605

Substance P and the neurokinin-1 receptor regulate electroencephalogram non-rapid eye movement sleep slow-wave activity locally

PubMed Central

Zielinski, Mark R.; Karpova, Svetlana A.; Yang, Xiaomei; Gerashchenko, Dmitry

2014-01-01

The neuropeptide substance P is an excitatory neurotransmitter produced by various cells including neurons and microglia that is involved in regulating inflammation and cerebral blood flow—functions that affect sleep and slow-wave activity (SWA). Substance P is the major ligand for the neurokinin-1 receptor (NK-1R), which is found throughout the brain including the cortex. The NK-1R is found on sleep-active cortical neurons expressing neuronal nitric oxide synthase whose activity is associated with SWA. We determined the effects of local cortical administration of a NK-1R agonist (substance P-fragment 1, 7) and a NK-1R antagonist (CP96345) on sleep and SWA in mice. The NK-1R agonist significantly enhanced SWA for several hours when applied locally to the cortex of the ipsilateral hemisphere as the electroencephalogram (EEG) electrode but not after application to the contralateral hemisphere when compared to saline vehicle control injections. In addition, a significant compensatory reduction in SWA was found after the NK-1R agonist-induced enhancements in SWA. Conversely, injections of the NK-1R antagonist into the cortex of the ipsilateral hemisphere of the EEG electrode attenuated SWA compared to vehicle injections but this effect was not found after injections of the NK-1R antagonist into contralateral hemisphere as the EEG electrode. Non-rapid eye movement sleep and rapid eye movement sleep duration responses after NK-1R agonist and antagonist injections were not significantly different from the responses to the vehicle. Our findings indicate that the substance P and the NK-1R are involved in regulating SWA locally. PMID:25301750

Differential requirements for the Ets transcription factor Elf-1 in the development of NKT cells and NK cells

PubMed Central

Choi, Hak-Jong; Geng, Yanbiao; Cho, Hoonsik; Li, Sha; Giri, Pramod Kumar; Felio, Kyrie

2011-01-01

E26 Transformation specific (Ets) family transcription factors control the expression of a large number of genes regulating hematopoietic cell development and function. Two such transcription factors, Ets-1 and myeloid Elf-1–like factor (MEF), have been shown to play critical roles in both natural killer (NK)– and NKT-cell development, but not in the development of conventional T cells. In this study, we address the role of E74-like factor 1 (Elf-1), another Ets family transcription factor that is closely related to MEF but divergent from Ets-1, in NK- and NKT-cell development using Elf-1–deficient (Elf-1−/−) mice. Whereas the proportion of NK cells in Elf-1−/− mice was normal, the proportion of NKT cells was significantly reduced in the thymus and periphery of Elf-1−/− mice compared with wild-type (WT) mice. Although Ets-1–deficient mice lack NKT cells altogether, Elf-1−/− mice exhibited only a partial block in NKT-cell development caused by a cell-intrinsic defect in the selection, survival, and maturation of NKT cells. In addition, residual NKT cells found in Elf-1−/− mice produced less cytokine upon antigen stimulation compared with WT NKT cells. Our data demonstrate that Elf-1 plays an important and nonredundant role in the development and function of NKT cells, but is not involved in NK-cell development. PMID:21148815

CXCR6 marks a novel subset of T-betloEomeshi natural killer cells residing in human liver

PubMed Central

Stegmann, Kerstin A.; Robertson, Francis; Hansi, Navjyot; Gill, Upkar; Pallant, Celeste; Christophides, Theodoros; Pallett, Laura J.; Peppa, Dimitra; Dunn, Claire; Fusai, Giuseppe; Male, Victoria; Davidson, Brian R.; Kennedy, Patrick; Maini, Mala K.

2016-01-01

Natural killer cells (NK) are highly enriched in the human liver, where they can regulate immunity and immunopathology. We probed them for a liver-resident subset, distinct from conventional bone-marrow-derived NK. CXCR6+ NK were strikingly enriched in healthy and diseased liver compared to blood (p < 0.0001). Human hepatic CXCR6+ NK had an immature phenotype (predominantly CD56brightCD16−CD57−), and expressed the tissue-residency marker CD69. CXCR6+ NK produced fewer cytotoxic mediators and pro-inflammatory cytokines than the non-liver-specific CXCR6− fraction. Instead CXCR6+ NK could upregulate TRAIL, a key death ligand in hepatitis pathogenesis. CXCR6 demarcated liver NK into two transcriptionally distinct populations: T-bethiEomeslo(CXCR6−) and T-betloEomeshi(CXCR6+); the latter was virtually absent in the periphery. The small circulating CXCR6+ subset was predominantly T-bethiEomeslo, suggesting its lineage was closer to CXCR6− peripheral than CXCR6+ liver NK. These data reveal a large subset of human liver-resident T-betloEomeshi NK, distinguished by their surface expression of CXCR6, adapted for hepatic tolerance and inducible anti-viral immunity. PMID:27210614

CXCR6 marks a novel subset of T-bet(lo)Eomes(hi) natural killer cells residing in human liver.

PubMed

Stegmann, Kerstin A; Robertson, Francis; Hansi, Navjyot; Gill, Upkar; Pallant, Celeste; Christophides, Theodoros; Pallett, Laura J; Peppa, Dimitra; Dunn, Claire; Fusai, Giuseppe; Male, Victoria; Davidson, Brian R; Kennedy, Patrick; Maini, Mala K

2016-05-23

Natural killer cells (NK) are highly enriched in the human liver, where they can regulate immunity and immunopathology. We probed them for a liver-resident subset, distinct from conventional bone-marrow-derived NK. CXCR6+ NK were strikingly enriched in healthy and diseased liver compared to blood (p < 0.0001). Human hepatic CXCR6+ NK had an immature phenotype (predominantly CD56(bright)CD16-CD57-), and expressed the tissue-residency marker CD69. CXCR6+ NK produced fewer cytotoxic mediators and pro-inflammatory cytokines than the non-liver-specific CXCR6- fraction. Instead CXCR6+ NK could upregulate TRAIL, a key death ligand in hepatitis pathogenesis. CXCR6 demarcated liver NK into two transcriptionally distinct populations: T-bet(hi)Eomes(lo)(CXCR6-) and T-bet(lo)Eomes(hi)(CXCR6+); the latter was virtually absent in the periphery. The small circulating CXCR6+ subset was predominantly T-bet(hi)Eomes(lo), suggesting its lineage was closer to CXCR6- peripheral than CXCR6+ liver NK. These data reveal a large subset of human liver-resident T-bet(lo)Eomes(hi) NK, distinguished by their surface expression of CXCR6, adapted for hepatic tolerance and inducible anti-viral immunity.

Cystatin F Affects Natural Killer Cell Cytotoxicity

PubMed Central

Perišić Nanut, Milica; Sabotič, Jerica; Švajger, Urban; Jewett, Anahid; Kos, Janko

2017-01-01

Cystatin F is a cysteine peptidase inhibitor which, unlike other cystatin family members, is targeted to endosomal/lysosomal compartments. It is synthesized as an inactive disulfide-linked dimer which is then converted to an active monomer by proteolytic cleavage of 15 N-terminal residues. Cystatin F has been suggested to regulate the cytotoxicity of natural killer (NK) cells by inhibiting the major granzyme convertases, cathepsins C and H. To test this hypothesis, we prepared variants of cystatin F and analyzed their uptake, subcellular trafficking, and peptidase inhibition, as well as their impact on the cytotoxicity of NK-92 cells and primary NK cells. The N-glycosylation pattern is responsible for the secretion, uptake, and subcellular sorting of cystatin F in HeLa and Hek293 cells, whereas the legumain binding site had no effect on these processes. Active, N-terminally truncated, monomeric cystatin F can also be internalized by recipient cells and targeted to endo/lysosomes, affecting also cells lacking the activating peptidase. Cystatin F mutants capable of cell internalization and trafficking through the endo/lysosomal pathway significantly decreased cathepsin C and H activities, both in situ, following transfection and in trans, using conditioned media. Further, incubation of IL-2 stimulated NK-92 and primary NK cells with full-length and N-terminally truncated cystatin F mutants led to suppression of their granule-mediated cytotoxicity. This effect was most significant with the N-terminally truncated mutants. These results suggest that cystatin F can be an important mediator within tumor microenvironment affecting the cytotoxicity of NK cells and consequently antitumor immune response. PMID:29180998

Endothelin-converting enzyme 1 degrades neuropeptides in endosomes to control receptor recycling.

PubMed

Roosterman, Dirk; Cottrell, Graeme S; Padilla, Benjamin E; Muller, Laurent; Eckman, Christopher B; Bunnett, Nigel W; Steinhoff, Martin

2007-07-10

Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with beta-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide-receptor-arrestin complex is sequestered into endosomes. Although dissociation of beta-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase endothelin-converting enzyme 1 (ECE-1), determines stability of the peptide-receptor-arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK1R) induced membrane translocation of beta-arrestins followed by trafficking of the SP-NK1R-beta-arrestin complex to early endosomes containing ECE-1a-d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; beta-arrestins returned to the cytosol, and the NK1R, freed from beta-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK1R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK3R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide-receptor-arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control beta-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.

Peripheral blood TIM-3 positive NK and CD8+ T cells throughout pregnancy: TIM-3/galectin-9 interaction and its possible role during pregnancy.

PubMed

Meggyes, Matyas; Miko, Eva; Polgar, Beata; Bogar, Barbara; Farkas, Balint; Illes, Zsolt; Szereday, Laszlo

2014-01-01

The T-cell immunoglobulin and mucin domain (TIM) family is a relatively newly described group of molecules with a conserved structure and important immunological functions. Identification of Galectin-9 as a ligand for TIM-3 has established the Galectin-9/TIM-3 pathway as an important negative regulator of Th1 immunity and tolerance induction. Data about the TIM-3/Gal-9 pathway in the pathogenesis of human diseases is emerging, but their possible role during human pregnancy is not precisely known. The aim of our study was to investigate the number, phenotype and functional activity of TIM-3+ peripheral blood mononuclear cells during healthy human pregnancy. 57 healthy pregnant women [first trimester (n = 16); second trimester (n = 19); third trimester (n = 22)] and 30 non-pregnant controls were enrolled in the study. We measured the surface expression of TIM-3 by cytotoxic T cells, NK cells and NK cell subsets as well as Galectin-9 expression by regulatory T cells by flow cytometry. We analyzed the cytokine production and cytotoxicity of TIM3+ and TIM3- CD8 T and NK cells obtained from non-pregnant and healthy pregnant women at different stages of pregnancy by flow cytometry. Serum Galectin-9 levels were measured by ELISA. Our results show that the numbers of peripheral NK and cytotoxic T cells and their TIM-3 expression do not change between the first, second and third trimesters of pregnancy. Compared to non-pregnant individuals, regulatory T cells show higher level of Galectin-9 expression as pregnancy proceeds, which is in line with the level of Galectin-9 in the patients sera. Cytotoxic T cells, NK cells and NK cell subsets expressing TIM-3 molecule show altered cytokine production and cytotoxicity during pregnancy compared to non-pregnant individuals. Our results indicate that Galectin-9 expressing regulatory T cells, TIM-3+ cytotoxic T cells and NK cells could play an important role in the maintenance of healthy pregnancy.

Liposomes loaded with a STING pathway ligand, cyclic di-GMP, enhance cancer immunotherapy against metastatic melanoma.

PubMed

Nakamura, Takashi; Miyabe, Hiroko; Hyodo, Mamoru; Sato, Yusuke; Hayakawa, Yoshihiro; Harashima, Hideyoshi

2015-10-28

Malignant melanomas escape immunosurveillance via the loss/down-regulation of MHC-I expression. Natural killer (NK) cells have the potential to function as essential effector cells for eliminating melanomas. Cyclic di-GMP (c-di-GMP), a ligand of the stimulator of interferon genes (STING) signal pathway, can be thought of as a new class of adjuvant against cancer. However, it is yet to be tested, because technologies for delivering c-di-GMP to the cytosol are required. Herein, we report that c-di-GMP efficiently activates NK cells and induces antitumor effects against malignant melanomas when loaded in YSK05 lipid containing liposomes, by assisting in the efficient delivery of c-di-GMP to the cytosol. The intravenous administration of c-di-GMP encapsulated within YSK05-liposomes (c-di-GMP/YSK05-Lip) into mice efficiently induced the production of type I interferon (IFN) as well as the activation of NK cells, resulting in a significant antitumor effect in a lung metastasis mouse model using B16-F10. This antitumor effect was dominated by NK cells. The infiltration of NK cells was observed in the lungs with B16-F10 melanomas. These findings indicate that the c-di-GMP/YSK05-Lip induces MHC-I non-restricted antitumor immunity mediated by NK cells. Consequently, c-di-GMP/YSK05-Lip represents a potentially new adjuvant system for use in immunotherapy against malignant melanomas. Copyright © 2015 Elsevier B.V. All rights reserved.

Clinical impact of the NKp30/B7-H6 axis in high-risk neuroblastoma patients.

PubMed

Semeraro, Michaela; Rusakiewicz, Sylvie; Minard-Colin, Véronique; Delahaye, Nicolas F; Enot, David; Vély, Frédéric; Marabelle, Aurélien; Papoular, Benjamin; Piperoglou, Christelle; Ponzoni, Mirco; Perri, Patrizia; Tchirkov, Andrei; Matta, Jessica; Lapierre, Valérie; Shekarian, Tala; Valsesia-Wittmann, Sandrine; Commo, Frédéric; Prada, Nicole; Poirier-Colame, Vichnou; Bressac, Brigitte; Cotteret, Sophie; Brugieres, Laurence; Farace, Françoise; Chaput, Nathalie; Kroemer, Guido; Valteau-Couanet, Dominique; Zitvogel, Laurence

2015-04-15

The immunosurveillance mechanisms governing high-risk neuroblastoma (HR-NB), a major pediatric malignancy, have been elusive. We identify a potential role for natural killer (NK) cells, in particular the interaction between the NK receptor NKp30 and its ligand, B7-H6, in the metastatic progression and survival of HR-NB after myeloablative multimodal chemotherapy and stem cell transplantation. NB cells expressing the NKp30 ligand B7-H6 stimulated NK cells in an NKp30-dependent manner. Serum concentration of soluble B7-H6 correlated with the down-regulation of NKp30, bone marrow metastases, and chemoresistance, and soluble B7-H6 contained in the serum of HR-NB patients inhibited NK cell functions in vitro. The expression of distinct NKp30 isoforms affecting the polarization of NK cell functions correlated with 10-year event-free survival in three independent cohorts of HR-NB in remission from metastases after induction chemotherapy (n = 196, P < 0.001), adding prognostic value to known risk factors such as N-Myc amplification and age >18 months. We conclude that the interaction between NKp30 and B7-H6 may contribute to the fate of NB patients and that both the expression of NKp30 isoforms on circulating NK cells and the concentration of soluble B7-H6 in the serum may be clinically useful as biomarkers for risk stratification. Copyright © 2015, American Association for the Advancement of Science.

Expression of killer inhibitory receptors on cytotoxic cells from HIV-1-infected individuals

PubMed Central

Galiani, M D; Aguado, E; Tarazona, R; Romero, P; Molina, I; Santamaria, M; Solana, R; PeñA, J

1999-01-01

Dysfunction of cytotoxic activity of T and natural killer (NK) lymphocytes is a main immunological feature in patients with AIDS, but its basis are not well understood. It has been recently described that T and NK cell-mediated cytotoxicity can be regulated by HLA killer inhibitory receptors (KIR). In this work, we have determined on cytotoxic T cells and NK cells from HIV-1-infected individuals the expression of the following KIR molecules: p58, p70, and ILT2 (immunoglobulin-like family KIR) as well as CD94 and NKG2A (C-lectin-type family KIR). With some exceptions, no significant changes were found on the expression of immunoglobulin-like KIR in either CD8+ or CD56+ cells. Interestingly, the percentages of CD8+ and CD56+ cells expressing CD94 were significantly increased in these individuals. We also show that, in vitro, IL-10 up-regulates CD94 expression on CD8+ and CD56+ cells obtained from normal individuals, suggesting that the augmented expression observed in HIV-infected individuals could be related to the high levels of IL-10 previously described in HIV-1-infected individuals. PMID:10193420

Diversification of both KIR and NKG2 natural killer cell receptor genes in macaques - implications for highly complex MHC-dependent regulation of natural killer cells.

PubMed

Walter, Lutz; Petersen, Beatrix

2017-02-01

The killer immunoglobulin-like receptors (KIR) as well as their MHC class I ligands display enormous genetic diversity and polymorphism in macaque species. Signals resulting from interaction between KIR or CD94/NKG2 receptors and their cognate MHC class I proteins essentially regulate the activity of natural killer (NK) cells. Macaque and human KIR share many features, such as clonal expression patterns, gene copy number variations, specificity for particular MHC class I allotypes, or epistasis between KIR and MHC class I genes that influence susceptibility and resistance to immunodeficiency virus infection. In this review article we also annotated publicly available rhesus macaque BAC clone sequences and provide the first description of the CD94-NKG2 genomic region. Besides the presence of genes that are orthologous to human NKG2A and NKG2F, this region contains three NKG2C paralogues. Hence, the genome of rhesus macaques contains moderately expanded and diversified NKG2 genes in addition to highly diversified KIR genes. The presence of two diversified NK cell receptor families in one species has not been described before and is expected to require a complex MHC-dependent regulation of NK cells. © 2016 John Wiley & Sons Ltd.

NK cell-based immunotherapy for malignant diseases

PubMed Central

Cheng, Min; Chen, Yongyan; Xiao, Weihua; Sun, Rui; Tian, Zhigang

2013-01-01

Natural killer (NK) cells play critical roles in host immunity against cancer. In response, cancers develop mechanisms to escape NK cell attack or induce defective NK cells. Current NK cell-based cancer immunotherapy aims to overcome NK cell paralysis using several approaches. One approach uses expanded allogeneic NK cells, which are not inhibited by self histocompatibility antigens like autologous NK cells, for adoptive cellular immunotherapy. Another adoptive transfer approach uses stable allogeneic NK cell lines, which is more practical for quality control and large-scale production. A third approach is genetic modification of fresh NK cells or NK cell lines to highly express cytokines, Fc receptors and/or chimeric tumor-antigen receptors. Therapeutic NK cells can be derived from various sources, including peripheral or cord blood cells, stem cells or even induced pluripotent stem cells (iPSCs), and a variety of stimulators can be used for large-scale production in laboratories or good manufacturing practice (GMP) facilities, including soluble growth factors, immobilized molecules or antibodies, and other cellular activators. A list of NK cell therapies to treat several types of cancer in clinical trials is reviewed here. Several different approaches to NK-based immunotherapy, such as tissue-specific NK cells, killer receptor-oriented NK cells and chemically treated NK cells, are discussed. A few new techniques or strategies to monitor NK cell therapy by non-invasive imaging, predetermine the efficiency of NK cell therapy by in vivo experiments and evaluate NK cell therapy approaches in clinical trials are also introduced. PMID:23604045

THE AP-2 CLATHRIN ADAPTOR MEDIATES ENDOCYTOSIS OF AN INHIBITORY KILLER CELL Ig-LIKE RECEPTOR (KIR) IN HUMAN NK CELLS1

PubMed Central

Purdy, Amanda K.; Alvarez-Arias, Diana A.; Oshinsky, Jennifer; James, Ashley M.; Serebriiskii, Ilya; Campbell, Kerry S.

2014-01-01

Stable surface expression of human inhibitory killer cell immunoglobulin-like receptors (KIR) is critical for controlling NK cell function and maintaining NK cell tolerance toward normal MHC-I+ cells. Our recent experiments, however, have found that antibody-bound KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the μ2 subunit of the AP-2 clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/μ2 interaction, either by mutation of the ITIM tyrosines in 3DL1 or mutation of μ2, significantly diminished endocytosis and increased surface expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is diminished upon antibody engagement with the receptor, as compared to untreated cells. Thus, we have identified AP-2-mediated endocytosis as a mechanism regulating the surface levels of inhibitory KIR though their ITIM domains. Based upon our results, we propose a model in which non-engaged KIR are internalized by this mechanism, whereas engagement with MHC-I ligand would diminish AP-2 binding, thereby prolonging stable receptor surface expression and promoting inhibitory function. Furthermore, this ITIM-mediated mechanism may similarly regulate the surface expression of other inhibitory immune receptors. PMID:25238755

The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells.

PubMed

Paolino, Magdalena; Choidas, Axel; Wallner, Stephanie; Pranjic, Blanka; Uribesalgo, Iris; Loeser, Stefanie; Jamieson, Amanda M; Langdon, Wallace Y; Ikeda, Fumiyo; Fededa, Juan Pablo; Cronin, Shane J; Nitsch, Roberto; Schultz-Fademrecht, Carsten; Eickhoff, Jan; Menninger, Sascha; Unger, Anke; Torka, Robert; Gruber, Thomas; Hinterleitner, Reinhard; Baier, Gottfried; Wolf, Dominik; Ullrich, Axel; Klebl, Bert M; Penninger, Josef M

2014-03-27

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.

Imaging immune surveillance of individual natural killer cells confined in microwell arrays.

PubMed

Guldevall, Karolin; Vanherberghen, Bruno; Frisk, Thomas; Hurtig, Johan; Christakou, Athanasia E; Manneberg, Otto; Lindström, Sara; Andersson-Svahn, Helene; Wiklund, Martin; Önfelt, Björn

2010-11-12

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.

IFN-γ elevates airway hyper-responsiveness via up-regulation of neurokinin A/neurokinin-2 receptor signaling in a severe asthma model.

PubMed

Kobayashi, Minoru; Ashino, Shigeru; Shiohama, Yasuo; Wakita, Daiko; Kitamura, Hidemitsu; Nishimura, Takashi

2012-02-01

The adoptive transfer of OVA-specific Th1 cells into WT mice followed by OVA inhalation induces a significant elevation of airway hyper-responsiveness (AHR) with neutrophilia but not mucus hypersecretion. Here, we demonstrate that the airway inflammation model, pathogenically characterized as severe asthma, was partly mimicked by i.n. administration of IFN-γ. The administration of IFN-γ instead of Th1 cells caused AHR elevation but not neutrophilia, and remarkably induced neurokinin-2 receptor (NK2R) expression along with neurokinin A (NKA) production in the lung. To evaluate whether NKA/NK2R was involved in airway inflammation, we first investigated the role of NKA/NK2R-signaling in airway smooth muscle cells (ASMCs) in vitro. NK2R mRNA expression was significantly augmented in tracheal tube-derived ASMCs of WT mice but not STAT-1(-/-) mice after stimulation with IFN-γ. In addition, methacholine-mediated Ca(2+) influx into the ASMCs was significantly reduced in the presence of NK2R antagonist. Moreover, the NK2R antagonist strongly inhibited IFN-γ-dependent AHR elevation in vivo. Thus, these results demonstrated that IFN-γ directly acts on ASMCs to elevate AHR via the NKA/NK2R-signaling cascade. Our present findings suggested that NK2R-mediated neuro-immuno crosstalk would be a promising target for developing novel drugs in Th1-cell-mediated airway inflammation, including severe asthma. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CD27 natural killer cell subsets play different roles during the pre-onset stage of experimental autoimmune encephalomyelitis.

PubMed

Gao, Ming; Yang, Yan; Li, Daling; Ming, Bingxia; Chen, Huoying; Sun, Yan; Xiao, Yifan; Lai, Lin; Zou, Huijuan; Xu, Yong; Xiong, Ping; Tan, Zheng; Gong, Feili; Zheng, Fang

2016-08-01

NK cells participate in the development of human multiple sclerosis (MS) and mouse experimental autoimmune encephalomyelitis (EAE), but the roles of different NK cell subsets in disease onset remain poorly understood. In this study, murine NK cells were divided into CD27(high) and CD27(low/-) subsets. The CD27(high) subset was decreased and the CD27(low/-) subset was increased in lymphoid organs during the pre-onset stage of EAE. Compared with the counterpart in naïve mice, the CD27(high) subset showed lower expression of Ly49D, Ly49H and NKG2D, and less production of IFN-γ, whereas the CD27(low/-) subset showed similar expression of the above mentioned surface receptors but higher cytotoxic activity in EAE mice. Compared with the CD27(high) subset, the CD27(low/-) subset exhibited increased promotion of DC maturation and no significant inhibition of T cells proliferation and Th17 cells differentiation in vitro Additionally, adoptive transfer of the CD27(low/-) subset, but not the CD27(high) subset, exacerbated the severity of EAE. Collectively, our data suggest the CD27 NK cell subsets play different roles in controlling EAE onset, which provide a new understanding for the regulation of NK cell subsets in early autoimmune disease. © The Author(s) 2016.

Human Cytomegalovirus UL18 Utilizes US6 for Evading the NK and T-Cell Responses

PubMed Central

Kim, Youngkyun; Park, Boyoun; Cho, Sunglim; Shin, Jinwook; Cho, Kwangmin; Jun, Youngsoo; Ahn, Kwangseog

2008-01-01

Human cytomegalovirus (HCMV) US6 glycoprotein inhibits TAP function, resulting in down-regulation of MHC class I molecules at the cell surface. Cells lacking MHC class I molecules are susceptible to NK cell lysis. HCMV expresses UL18, a MHC class I homolog that functions as a surrogate to prevent host cell lysis. Despite a high level of sequence and structural homology between UL18 and MHC class I molecules, surface expression of MHC class I, but not UL18, is down regulated by US6. Here, we describe a mechanism of action by which HCMV UL18 avoids attack by the self-derived TAP inhibitor US6. UL18 abrogates US6 inhibition of ATP binding by TAP and, thereby, restores TAP-mediated peptide translocation. In addition, UL18 together with US6 interferes with the physical association between MHC class I molecules and TAP that is required for optimal peptide loading. Thus, regardless of the recovery of TAP function, surface expression of MHC class I molecules remains decreased. UL18 represents a unique immune evasion protein that has evolved to evade both the NK and the T cell immune responses. PMID:18688275

Impact of C-rel inhibition of cord blood-derived B-, T-, and NK cells.

PubMed

Fallahi, Shirin; Mohammadi, Seyede Momeneh; Tayefi Nasrabadi, Hamid; Alihemmati, Alireza; Samadi, Naser; Gholami, Sanaz; Shanehbandi, Dariush; Nozad Charoudeh, Hojjatollah

2017-12-01

The c-Rel transcription factor is a unique member of the nuclear factor (NF)-κB family that has a role in curtailing the proliferation, differentiation, cytokine production, and overall activity of B- and T-cells. In addition, c-Rel is a key regulator of apoptosis in that it influences the expression of anti-apoptotic genes such as Bcl-2 and Bcl-xL; conversely, inhibition of c-Rel increases cell apoptosis. To better understand the relationship between c-Rel expression and effects on B- and T-cell expansion, the current study evaluated c-Rel expression in cord blood mononuclear cells. This particular source was selected as cord blood is an important source of cells used for transplantation and immunotherapy, primarily in treating leukemias. As stem cell factor (SCF) and FLT3 are important agents for hematopoietic stem cell expansion, and cytokines like interleukin (IL)-2, -7, and -15 are essential for T- and B- (and also NK) cell development and proliferation, the current study evaluated c-Rel expression in cord blood mononuclear cells and CD34 +  cells, as well as effects on B-, T-, and NK cells associated with alterations in c-Rel expression, using flow cytometry and PCR. The results showed c-Rel expression increased among cells cultured in the presence of SCF and FLT3 but was reduced when IL-2, IL-7, and IL-15 were used all together. Further, inhibition of c-Rel expression by siRNA reduced cord blood-derived B-, T-, and NK cell differentiation and expansion. These results indicated that with cells isolated from cord blood, c-Rel has an important role in B-, T-, and NK cell differentiation and, further, that agents (select cytokines/growth factors) that could impact on its expression might not only affect immune cell profiles in a host but could potentially also limit apoptotic activities in (non-)immune cells in that host. In the context of cancer (immuno)therapy, in particular, when cord blood is used an important source in stem cell transplantation in leukemia patients, such down-regulating changes in c-Rel levels could be counter-productive.

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells

PubMed Central

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach. PMID:25949869

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells.

PubMed

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach.

DNA demethylation and histone H3K9 acetylation determine the active transcription of the NKG2D gene in human CD8+ T and NK cells

PubMed Central

Fernández-Sánchez, Alba; Baragaño Raneros, Aroa; Carvajal Palao, Reyes; Sanz, Ana B.; Ortiz, Alberto; Ortega, Francisco; Suárez-Álvarez, Beatriz; López-Larrea, Carlos

2013-01-01

The human activating receptor NKG2D is mainly expressed by NK, NKT, γδ T and CD8+ T cells and, under certain conditions, by CD4+ T cells. This receptor recognizes a diverse family of ligands (MICA, MICB and ULBPs 1–6) leading to the activation of effector cells and triggering the lysis of target cells. The NKG2D receptor-ligand system plays an important role in the immune response to infections, tumors, transplanted graft and autoantigens. Elucidation of the regulatory mechanisms of NKG2D is therefore essential for therapeutic purposes. In this study, we speculate whether epigenetic mechanisms, such as DNA methylation and histone acetylation, participate in NKG2D gene regulation in T lymphocytes and NK cells. DNA methylation in the NKG2D gene was observed in CD4+ T lymphocytes and T cell lines (Jurkat and HUT78), while this gene was unmethylated in NKG2D-positive cells (CD8+ T lymphocytes, NK cells and NKL cell line) and associated with high levels of histone H3 lysine 9 acetylation (H3K9Ac). Treatment with the histone acetyltransferase (HAT) inhibitor curcumin reduces H3K9Ac levels in the NKG2D gene, downregulates NKG2D transcription and leads to a marked reduction in the lytic capacity of NKG2D-mediated NKL cells. These findings suggest that differential NKG2D expression in the different cell subsets is regulated by epigenetic mechanisms and that its modulation by epigenetic treatments might provide a new strategy for treating several pathologies. PMID:23235109

Adaptive NKG2C+CD57+ Natural Killer Cell and Tim-3 Expression During Viral Infections

PubMed Central

Kared, Hassen; Martelli, Serena; Tan, Shu Wen; Simoni, Yannick; Chong, Meng Li; Yap, Siew Hwei; Newell, Evan W.; Pender, Sylvia L. F.; Kamarulzaman, Adeeba; Rajasuriar, Reena; Larbi, Anis

2018-01-01

Repetitive stimulation by persistent pathogens such as human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV) induces the differentiation of natural killer (NK) cells. This maturation pathway is characterized by the acquisition of phenotypic markers, CD2, CD57, and NKG2C, and effector functions—a process regulated by Tim-3 and orchestrated by a complex network of transcriptional factors, involving T-bet, Eomes, Zeb2, promyelocytic leukemia zinc finger protein, and Foxo3. Here, we show that persistent immune activation during chronic viral co-infections (HCMV, hepatitis C virus, and HIV) interferes with the functional phenotype of NK cells by modulating the Tim-3 pathway; a decrease in Tim-3 expression combined with the acquisition of inhibitory receptors skewed NK cells toward an exhausted and cytotoxic phenotype in an inflammatory environment during chronic HIV infection. A better understanding of the mechanisms underlying NK cell differentiation could aid the identification of new immunological targets for checkpoint blockade therapies in a manner that is relevant to chronic infection and cancer. PMID:29731749

Cryptococcus Neoformans Modulates Extracellular Killing by Neutrophils

PubMed Central

Qureshi, Asfia; Grey, Angus; Rose, Kristie L.; Schey, Kevin L.; Del Poeta, Maurizio

2011-01-01

We recently established a key role for host sphingomyelin synthase (SMS) in regulating the killing activity of neutrophils against Cryptococcus neoformans. In this paper, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and natural killer (NK) cells (Tgε26 mice). To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in vitro and in vivo. We show that unlike Candida albicans, neither the presence nor the capsule size of C. neoformans cells have any effect on neutrophil viability. Interestingly, melanized C. neoformans cells totally abrogated the killing activity of neutrophils. We monitored how exposure of neutrophils to C. neoformans cells would interfere with any further killing activity of the conditioned medium and found that pre-incubation with live but not “heat-killed” fungal cells significantly inhibits further killing activity of the medium. We then studied whether activation of SMS at the site of C. neoformans infection is dependent on T and NK cells. Using matrix-assisted laser desorption–ionization tissue imaging in infected lung we found that similar to previous observations in the isogenic wild-type CBA/J mice, SM 16:0 levels are significantly elevated at the site of infection in mice lacking T and NK cells, but only at early time points. This study highlights that C. neoformans may negatively regulate the killing activity of neutrophils and that SMS activation in neutrophils appears to be partially independent of T and/or NK cells. PMID:21960987

Clinical-grade purification of natural killer cells in haploidentical hematopoietic stem cell transplantation.

PubMed

Meyer-Monard, Sandrine; Passweg, Jakob; Siegler, Uwe; Kalberer, Christian; Koehl, Ulrike; Rovó, Alicia; Halter, Jörg; Stern, Martin; Heim, Dominik; Alois Gratwohl, Johannes Rischewski; Tichelli, André

2009-02-01

Because of a high risk of graft-versus-host disease (GVHD), donor lymphocyte infusions with unmodified lymphapheresis products are not used after haploidentical hematopoietic stem cell transplantation. Natural killer (NK) cells have antitumor activity and may consolidate engraftment without inducing GVHD. Production of NK cells under good manufacturing practice (GMP) conditions in a sufficient number is difficult. Twenty-four apheresis procedures and subsequent NK-cell enrichment from 14 haploidentical donors were performed. NK-cell enrichment was performed using a GMP suitable immunomagnetic procedure. Factors influencing the NK-cell recovery, purity, and NK-cell dose were analyzed. A median number of 4.9 x 10(8) NK cells were obtained and median NK-cell recovery was 58 percent. Median T-cell depletion was 4.32 log. The absolute NK-cell number in the final product after processing significantly correlated with the preharvest NK-cell content of the peripheral blood (p = 0.002, r = 0.867). The NK-cell recovery was inversely correlated to the absolute NK-cell number in the apheresis product (p = 0.01, r = -0.51). The NK-cell dose per kg of body weight of the patient was inversely correlated to the weight of the patient (p = 0.007, r = -0.533). Donors with a high NK-cell count in peripheral blood are likely to provide NK-cell products with the highest cell number. However, maximal NK-cell dose is limited and high NK-cell doses may only be obtained for patients with a low body weight, making children and young adults the best candidates for NK-cell therapy.

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

PubMed

Lupatov, A Yu; Kim, Ya S; Bystrykh, O A; Vakhrushev, I V; Pavlovich, S V; Yarygin, K N; Sukhikh, G T

2017-02-01

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

Potentiating NK cell activity by combination of Rosuvastatin and Difluoromethylornithine for effective chemopreventive efficacy against Colon Cancer

PubMed Central

Janakiram, Naveena B.; Mohammed, Altaf; Bryant, Taylor; Zhang, Yuting; Brewer, Misty; Duff, Ashley; Biddick, Laura; Singh, Anil; Lightfoot, Stan; Steele, Vernon E; Rao, Chinthalapally V.

2016-01-01

Colorectal cancer (CRC) is the second highest cause of cancer-related deaths. A successful strategy to improve chemopreventive efficacies is by down-regulating tumor polyamines and enhancing NK cell activities. Colonic carcinogenesis was induced by azoxymethane (AOM) in male F344 rats. Eight weeks after AOM treatment, animals were fed diets containing Rosuvastatin and difluromethylornithine (DFMO) individually and in combination for 40 weeks. Both agents showed significant suppression of adenocarcinoma multiplicity and incidence with no toxicity compared to untreated rats. Low-dose Rosuvastatin plus DFMO suppressed colon adenocarcinoma multiplicity by 76% compared to low-dose Rosuvastatin (29%) and DFMO (46%), suggesting additive efficacy. Furthermore, low-dose combination caused a delay in colonic adenocarcinoma progression. DFMO, Rosuvastatin and/or combinations significantly decreased polyamine content and increased intra-tumoral NK cells expressing perforin plus IFN-γ compared to untreated colon tumors. Further ex-vivo analysis of splenic NK cells exposed to DFMO, Rosuvastatin or combination resulted in an increase of NKs with perforin expression. This is the first report on Rosuvastatin alone or combination strategy using clinically relevant statin plus DFMO doses which shows a significant suppression of colon adenocarcinomas, and their potential in increasing functional NK cells. This strategy has potential for further testing in high risk individuals for colon cancer. PMID:27841323

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Guojun

Staphylococcal enterotoxin C2 (SEC2), a member of bacterial superantigen, is one of the most potent known activators of T lymphocytes. With this property, SEC2 has already been used in clinic as a tumor immunotherapy agent in China. To increase the antitumor activity, a SEC2 mutant named ST-4 (GKVTG102-106WWH) with amino acid substitutions in T cell receptor (TCR)-binding domain was generated by site-directed mutagenesis, and the molecular mechanism of the enhanced antitumor activity was investigated. Results showed that ST-4 could activate much more Vβ 8.2 and 8.3 T cells and NK cells compared with SEC2, and exhibited significantly enhanced immunocyte stimulationmore » and antitumor activity in vitro. The synthetic peptide sequencing the residues of mutant TCR-binding domain could competitively inhibit the immunocyte stimulation activity of ST-4. Most importantly, ST-4 up-regulated granzyme B and perforin at both mRNA and protein levels. We also found that expression of proapoptotic proteins cytochrome c, BAX and activation of caspase-3, 9 was up-regulated, and antiapoptotic protein Bcl-xL was down-regulated in the treatment with either ST-4 or SEC2. When granzyme B inhibitor or perforin inhibitor is presented, tumor cell viability was significantly rescued. Taken together, we demonstrate that increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. These activated cells released up-regulated granzyme B and perforin, which induced the enhanced tumor cells apoptosis by mitochondrial apoptotic pathway, and ultimately led to enhanced tumor cell growth inhibition. ST-4 may be a promising candidate for antitumor clinic usage in future. - Highlights: • We obtained a SEC2 mutant ST-4 with enhanced superantigen and antitumor activity. • Increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. • Up-regulated GzmB and PRF1 in T cell by ST-4 induced enhanced tumor cells apoptosis. • Enhanced tumor cell apoptosis induced by ST-4 via mitochondrial apoptotic pathway.« less

Uterine activin receptor-like kinase 5 is crucial for blastocyst implantation and placental development

PubMed Central

Peng, Jia; Monsivais, Diana; You, Ran; Zhong, Hua; Pangas, Stephanie A.; Matzuk, Martin M.

2015-01-01

Members of the transforming growth factor β (TGF-β) superfamily are key regulators in most developmental and physiological processes. However, the in vivo roles of TGF-β signaling in female reproduction remain uncertain. Activin receptor-like kinase 5 (ALK5) is the major type 1 receptor for the TGF-β subfamily. Absence of ALK5 leads to early embryonic lethality because of severe defects in vascular development. In this study, we conditionally ablated uterine ALK5 using progesterone receptor-cre mice to define the physiological roles of ALK5 in female reproduction. Despite normal ovarian functions and artificial decidualization in conditional knockout (cKO) mice, absence of uterine ALK5 resulted in substantially reduced female reproduction due to abnormalities observed at different stages of pregnancy, including implantation defects, disorganization of trophoblast cells, fewer uterine natural killer (uNK) cells, and impairment of spiral artery remodeling. In our microarray analysis, genes encoding proteins involved in cytokine–cytokine receptor interactions and NK cell-mediated cytotoxicity were down-regulated in cKO decidua compared with control decidua. Flow cytometry confirmed a 10-fold decrease in uNK cells in cKO versus control decidua. According to these data, we hypothesize that TGF-β acts on decidual cells via ALK5 to induce expression of other growth factors and cytokines, which are key regulators in luminal epithelium proliferation, trophoblast development, and uNK maturation during pregnancy. Our findings not only generate a mouse model to study TGF-β signaling in female reproduction but also shed light on the pathogenesis of many pregnancy complications in human, such as recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction. PMID:26305969

Shaping of Natural Killer Cell Antitumor Activity by Ex Vivo Cultivation

PubMed Central

Granzin, Markus; Wagner, Juliane; Köhl, Ulrike; Cerwenka, Adelheid; Huppert, Volker; Ullrich, Evelyn

2017-01-01

Natural killer (NK) cells are a promising tool for the use in adoptive immunotherapy, since they efficiently recognize and kill tumor cells. In this context, ex vivo cultivation is an attractive option to increase NK cells in numbers and to improve their antitumor potential prior to clinical applications. Consequently, various strategies to generate NK cells for adoptive immunotherapy have been developed. Here, we give an overview of different NK cell cultivation approaches and their impact on shaping the NK cell antitumor activity. So far, the cytokines interleukin (IL)-2, IL-12, IL-15, IL-18, and IL-21 are used to culture and expand NK cells. The selection of the respective cytokine combination is an important factor that directly affects NK cell maturation, proliferation, survival, distribution of NK cell subpopulations, activation, and function in terms of cytokine production and cytotoxic potential. Importantly, cytokines can upregulate the expression of certain activating receptors on NK cells, thereby increasing their responsiveness against tumor cells that express the corresponding ligands. Apart from using cytokines, cocultivation with autologous accessory non-NK cells or addition of growth-inactivated feeder cells are approaches for NK cell cultivation with pronounced effects on NK cell activation and expansion. Furthermore, ex vivo cultivation was reported to prime NK cells for the killing of tumor cells that were previously resistant to NK cell attack. In general, NK cells become frequently dysfunctional in cancer patients, for instance, by downregulation of NK cell activating receptors, disabling them in their antitumor response. In such scenario, ex vivo cultivation can be helpful to arm NK cells with enhanced antitumor properties to overcome immunosuppression. In this review, we summarize the current knowledge on NK cell modulation by different ex vivo cultivation strategies focused on increasing NK cytotoxicity for clinical application in malignant diseases. Moreover, we critically discuss the technical and regulatory aspects and challenges underlying NK cell based therapeutic approaches in the clinics. PMID:28491060

[Electroacupuncture Intervention Enhances Splenic Natural Killer Cell Activity via Inhibiting Phosphorylation of ERK 5 in the Hypothalamus of Surgically Traumatized Rats].

PubMed

Chen, Yan; Li, Jing; Zhu, Ke-ying; Xiao, Sheng; Wang, Yan-qing; Wu, Gen-cheng; Wang, Jun

2015-06-01

To observe the effect of electroacupuncture (EA) on cytotoxic activity of splenic natural killer (NK) cells after surgical trauma via extracellular signal-regulated kinase (ERK) 5 pathway in the rats' hypothalamus, so as to explore its mechanism underlying improving immune disorders after surgery. Sprague-Dawley rats were randomly divided into the following 6 groups: control, trauma model, EA, sham EA, 4 nmol-BIX 02188 (an inhibitor for ERK 5 catalytic activity) and 20 nmol-BIX 02188 (n = 6 rats per group). The surgical trauma model was established by making a longitudinal incision (6 cm in length) along the median line of the back to expose the spinal column and another longitudinal incision along the abdominal median line. EA (2 Hz/15 Hz, 1 - 2 mA) was applied to bilateral "Zusanli" (ST 36) for 30 min immediately after surgery. For rats of the BIX groups, intra-lateral ventricular microinjection of BIX 02188 (10 µL, 4 nmol or 20 nmol, or saline for control rats) was conducted 30 min before the surgery. The expression level and protein of phosphorylated ERK 5 (p-ERK 5) and corticotropin-releasing factor (CRF) protein were measured by immunohistochemistry and Western blot, respectively. The cytotoxicity of splenic NK cells and the expression of splenic Perforin and Granzyme-B genes were measured by lactate dehydrogenase (LDH) release assay and real-time PCR, respectively. In comparison with the control group, hypothalamic p-ERK 5 immunoactivity, p-ERK 5 protein and CRF protein expression levels were significantly up-regulated in the model group (P<0. 01, P<0. 05), while splenic NK cell cytotoxicity and Perforin mRNA and Granzyme-B mRNA expression levels were notably down-regulated in the model group (P <0. 05, P < 0. 01). Following EA and administration of ERK 5 antagonist, the increased expression levels of p-ERK 5 immunoactivity in the EA group, and p-ERK 5 and CRF proteins in both EA and 20 nmol-BIX 02188 groups were obviously down-regulated (P<0. 05, P<0. 01), without changes in the sham EA and 4 nmol-BIX 02188 groups (P>0. 05) except the increased p-ERK 5 protein in the 4 nmol-BIX 02188 group. In addition, the down-regulated NK cell activity, Perforin mRNA and Granzyme-B mRNA expression levels were significantly reversed in the EA and 20 nmol-BIX 02188 groups (P<0. 05, P<0. 01). No significant differences were found between the EA group and 20 nmol-BIX 02188 group in down-regulating hypothalamic p-ERK 5 and CRF protein expression and up-regulating splenic NK cytotoxicity and Perforin and Granzyme-B gene expression (P>0. 05). EA can promote the cytotoxicity of splenic NK cells in surgical trauma rats, which may be closely associated with its functions in down-regulating trauma-induced activation of ERK 5 pathway and production of CRF in the hypothalamus.

Substance P Receptor Antagonist Suppresses Inflammatory Cytokine Expression in Human Disc Cells.

PubMed

Kepler, Christopher K; Markova, Dessislava Z; Koerner, John D; Mendelis, Joseph; Chen, Chiu-Ming; Vaccaro, Alexander R; Risbud, Makarand V; Albert, Todd J; Anderson, D Greg

2015-08-15

Laboratory study. To evaluate whether blockade of the Substance P (SP) NK1R attenuates its proinflammatory effect on human intervertebral disc cells (IVD), and to evaluate the signaling pathways associated with SP. SP and its receptors are expressed in human IVD cells, and cause upregulation of inflammatory mediators; however, the effects of blocking these receptors have not been studied in human IVD cells. Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were expanded in monolayer, and then suspended in alginate beads. The alginate beads were treated with culture medium first containing a high affinity NK1R antagonist (L-760735) at different concentrations, and then with medium containing both NK1R antagonist and SP at 2 concentrations. Ribonucleic acid was isolated and transcribed into cDNA. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate expression of interleukin (IL)-1β, IL-6, and IL-8. Western blot analysis was performed to examine levels of the phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65). The cells were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (PD98059), and p65 (SM7368) and then stimulated with SP. We detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R) in AF and NP cells. Treatment of disc cells with the NK1R antagonist was able to suppress expression of IL-1β, IL-6, and IL-8 in a dose-dependent manner. SP stimulation increased phosphorylation of p38-MAPK and ERK1/2, but not of NFκB p65. This indicates that p38-MAPK and ERK1/2 control SP-induced cytokine expression independently from NF-kB p65. Inhibition of p38 and ERK1/2 activation reduced SP-induced IL-6 production in human disc cells. NK1R is responsible for the proinflammatory effect of SP on IVD cells and this effect can be blocked by preventing binding of SP to NK1R. This study shows for the first time that SP mediates signaling in disc cells through NK1R and that SP activates the proinflammatory p38-MAPK and ERK1/2 pathways. 4.

Cytokine Expression, Natural Killer Cell Activation, and Phenotypic Changes in Lymphoid Cells from Rhesus Macaques during Acute Infection with Pathogenic Simian Immunodeficiency Virus

PubMed Central

Giavedoni, Luis D.; Velasquillo, M. Cristina; Parodi, Laura M.; Hubbard, Gene B.; Hodara, Vida L.

2000-01-01

We studied the innate and adaptive immune system of rhesus macaques infected with the virulent simian immunodeficiency virus isolate SIVmac251 by evaluating natural killer (NK) cell activity, cytokine levels in plasma, humoral and virological parameters, and changes in the activation markers CD25 (interleukin 2R [IL-2R] α chain), CD69 (early activation marker), and CD154 (CD40 ligand) in lymphoid cells. We found that infection with SIVmac251 induced the sequential production of interferon-α/β (IFN-α/β), IL-18, and IL-12. IFN-γ, IL-4, and granulocyte-macrophage colony-stimulating factor were undetected in plasma by the assays used. NK cell activity peaked at 1 to 2 weeks postinfection and paralleled changes in viral loads. Maximum expression of CD69 on CD3−CD16+ lymphocytes correlated with NK cytotoxicity during this period. CD25 expression, which is associated with proliferation, was static or slightly down-regulated in CD4+ T cells from both peripheral blood (PB) and lymph nodes (LN). CD69, which is normally present in LN CD4+ T cells and absent in peripheral blood leukocyte (PBL) CD4+ T cells, was down-regulated in LN CD4+ T cells and up-regulated in PBL CD4+ T cells immediately after infection. CD8+ T cells increased CD69 but not CD25 expression, indicating the activation of this cellular subset in PB and LN. Finally, CD154 was transiently up-regulated in PBL CD4+ T cells but not in LN CD4+ T cells. Levels of antibodies to SIV Gag and Env did not correlate with the level of activation of CD154, a critical costimulatory molecule for T-cell-dependent immunity. In summary, we present the first documented evidence that the innate immune system of rhesus macaques recognizes SIV infection by sequential production of proinflammatory cytokines and transient activation of NK cytotoxic activity. Additionally, pathogenic SIV induces drastic changes in the level of activation markers on T cells from different anatomic compartments. These changes involve activation in the absence of proliferation, indicating that activation-induced cell death may cause some of the reported increase in lymphocyte turnover during SIV infection. PMID:10644334

Activation of neurokinin-1 receptor by substance P inhibits melanogenesis in B16-F10 melanoma cells.

PubMed

Ping, Fengfeng; Shang, Jing; Zhou, Jia; Song, Jing; Zhang, Luyong

2012-12-01

Skin pigmentation plays a number of valuable roles and its regulation is a complex process that is controlled by different factors. Substance P (SP) regulates many biological functions, including neurogenic inflammation, pain, and stress. However, to date, the regulatory role of SP in the control of melanogenesis has not been elucidated. The present study was designed to investigate the effects of SP on melanogenesis and to elucidate its underlying mechanism(s). After treatment for 48 h in mouse B16-F10 melanoma cells, SP (1 and 10nM) significantly down-regulated tyrosinase activity and melanin content. Importantly, western blot analysis revealed the presence of neurokinin-1 receptor (NK-1 R) in B16-F10 cells and the activation of it after SP treatment. It was also found that preincubation with NK-1 receptor antagonist Spantide I could partially reversed SP-induced down-regulations of tyrosinase activity, melanin content and the expression of tyrosinase and tyrosinase-related protein 1. Furthermore, SP could remarkably inhibit the expressions of microphtalmia-associated transcription factor (MITF) and p-p38 MAPK and stimulated p-p70 S6K1. These effects could also be partially reversed by the pretreatment with Spantide I. These results collectively suggested that SP inhibited melanogenesis in B16-F10 cells, which might be attributed to the fact that SP induces the activation of NK-1 receptor, stimulates the phosphorylation of p70 S6K1 and inhibits that of p38 MAPK, decreases the tyrosinase and tyrosinase-related protein 1 expression through MITF, finally resulting in the suppression of melanogenesis. These observations in vitro indicated that the regulation of the SP/NK-1 receptor system might be a useful novel management for skin pigmentation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Substance P and the neurokinin-1 receptor expression in dog ileum with and without inflammation.

PubMed

Polidoro, Giulia; Giancola, Fiorella; Fracassi, Federico; Pietra, Marco; Bettini, Giuliano; Asti, Martina; Chiocchetti, Roberto

2017-10-01

In the gastrointestinal tract, the tachykinin Substance P (SP) is involved in motility, fluid and electrolyte secretion, and blood flow and regulation of immunoinflammatory response. SP exerts its biological activity on target cells by interacting mainly with the neurokinin-1 receptor (NK 1 R). The present study aims to quantify the percentage of SP-immunoreactive (SP-IR) enteric neurons and the density of SP-IR nerve fibers in the ileum of control dogs (CTRL-dogs; n=7) vs dogs with spontaneous ileal inflammation (INF-dogs; n=8). In addition, the percentage of enteric neurons bearing NK 1 R, and nitrergic neurons (nNOS-IR) expressing NK 1 R immunoreactivity were evaluated in both groups. The percentages of SP-IR neurons were similar in CTRL- and INF-dogs, in either the myenteric (MP) (15±8% vs. 16±7%, respectively) and submucosal plexus (SMP) (26±7% vs. 24±14%, respectively). In INF-dogs, the density of SP-IR mucosal nerve fibers showed a trend to decrease (P=0.07). Myenteric neurons of CTRL- and INF-dogs expressed similar percentages of NK 1 R-immunoreactivity (39±5% vs. 38±20%, respectively). Submucosal NK 1 R-IR neurons were occasionally observed in a CTRL-dog. MP nitrergic neurons bearing NK 1 R showed a trend to decrease in INF-dogs vs. CTRL- dogs (41±22% vs. 65±10%, respectively; P=0.11). In INF-dogs, muscle cells and immune cells overexpressed NK 1 R immunoreactivity. These findings should be taken as a warning for possible intestinal motility disorders, which might occur during administration of NK 1 R-antagonist drugs. Conversely, the strong expression of NK 1 R immunoreactivity observed in muscle and mucosal immune cells of inflamed tissues may provide a rationale for the use of NK 1 R antagonist drugs in the treatment of intestinal inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

ADCC employing an NK cell line (haNK) expressing the high affinity CD16 allele with avelumab, an anti-PD-L1 antibody.

PubMed

Jochems, Caroline; Hodge, James W; Fantini, Massimo; Tsang, Kwong Y; Vandeveer, Amanda J; Gulley, James L; Schlom, Jeffrey

2017-08-01

NK-92 cells, and their derivative, designated aNK, were obtained from a patient with non-Hodgkin lymphoma. Prior clinical studies employing adoptively transferred irradiated aNK cells have provided evidence of clinical benefit and an acceptable safety profile. aNK cells have now been engineered to express IL-2 and the high affinity (ha) CD16 allele (designated haNK). Avelumab is a human IgG1 anti-PD-L1 monoclonal antibody, which has shown evidence of clinical activity in a range of human tumors. Prior in vitro studies have shown that avelumab has the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of human tumor cells when combined with NK cells. In the studies reported here, the ability of avelumab to enhance the lysis of a range of human carcinoma cells by irradiated haNK cells via the ADCC mechanism is demonstrated; this ADCC is shown to be inhibited by anti-CD16 blocking antibody and by concanamycin A, indicating the use of the granzyme/perforin pathway in tumor cell lysis. Studies also show that while NK cells have the ability to lyse aNK or haNK cells, the addition of NK cells to irradiated haNK cells does not inhibit haNK-mediated lysis of human tumor cells, with or without the addition of avelumab. Avelumab-mediated lysis of tumor cells by irradiated haNK cells is also shown to be similar to that of NK cells bearing the V/V Fc receptor high affinity allele. These studies thus provide the rationale for the clinical evaluation of the combined use of avelumab with that of irradiated adoptively transferred haNK cells. © 2017 UICC.

Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

NASA Astrophysics Data System (ADS)

Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

2017-01-01

Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited.

Liver-resident NK cells and their potential functions.

PubMed

Peng, Hui; Sun, Rui

2017-09-18

Natural killer (NK) cells represent a heterogeneous population of innate lymphocytes with phenotypically and functionally distinct subsets. In particular, recent studies have identified a unique subset of NK cells residing within the liver that are maintained as tissue-resident cells, confer antigen-specific memory responses and exhibit different phenotypical and developmental characteristics compared with conventional NK (cNK) cells. These findings have encouraged researchers to uncover tissue-resident NK cells at other sites, and detailed analyses have revealed that these tissue-resident NK cells share many similarities with liver-resident NK cells and tissue-resident memory T cells. Here, we present a brief historical perspective on the discovery of liver-resident NK cells and discuss their relationship to cNK cells and other emerging NK cell subsets and their potential functions.Cellular &Molecular Immunology advance online publication, 18 September 2017; doi:10.1038/cmi.2017.72.

TGF-β1 Downregulates the Expression of CX3CR1 by Inducing miR-27a-5p in Primary Human NK Cells.

PubMed

Regis, Stefano; Caliendo, Fabio; Dondero, Alessandra; Casu, Beatrice; Romano, Filomena; Loiacono, Fabrizio; Moretta, Alessandro; Bottino, Cristina; Castriconi, Roberta

2017-01-01

Activity of human natural killer (NK) cells against cancer cells is deeply suppressed by TGF-β1, an immunomodulatory cytokine that is released and activated in the tumor microenvironment. Moreover, our previous data showed that TGF-β1 modifies the chemokine receptor repertoire of NK cells. In particular, it decreases the expression of CX 3 CR1 that drives these effectors toward peripheral tissues, including tumor sites. To identify possible mechanisms mediating chemokine receptors modulation, we analyzed the microRNA profile of TGF-β1-treated primary NK cells. The analysis pointed out miR-27a-5p as a possible modulator of CX 3 CR1. We demonstrated the functional interaction of miR-27a-5p with the 3' untranslated region (3'UTR) of CX 3 CR1 mRNA by two different experimental approaches: by the use of a luciferase assay based on a reporter construct containing the CX 3 CR1 3'UTR and by transfection of primary NK cells with a miR-27a-5p inhibitor. We also showed that the TGF-β1-mediated increase of miR-27a-5p expression is a consequence of miR-23a-27a-24-2 cluster induction. Moreover, we demonstrated that miR-27a-5p downregulates the surface expression of CX 3 CR1. Finally, we showed that neuroblastoma cells induced in resting NK cells a downregulation of the CX 3 CR1 expression that was paralleled by a significant increase of miR-27a-5p expression. Therefore, the present study highlights miR-27a-5p as a pivotal TGF-β1-induced regulator of CX 3 CR1 expression.

CD94/NKG2A inhibits NK cell activation by disrupting the actin network at the immunological synapse.

PubMed

Masilamani, Madhan; Nguyen, Connie; Kabat, Juraj; Borrego, Francisco; Coligan, John E

2006-09-15

An adequate immune response is the result of the fine balance between activation and inhibitory signals. The exact means by which inhibitory signals obviate activation signals in immune cells are not totally elucidated. Human CD94/NKG2A is an ITIM-containing inhibitory receptor expressed by NK cells and some CD8+ T cells that recognize HLA-E. We show that the engagement of this receptor prevents NK cell activation by disruption of the actin network and exclusion of lipid rafts at the point of contact with its ligand (inhibitory NK cell immunological synapse, iNKIS). CD94/NKG2A engagement leads to recruitment and activation of src homology 2 domain-bearing tyrosine phosphatase 1. This likely explains the observed dephosphorylation of guanine nucleotide exchange factor and regulator of actin, Vav1, as well as ezrin-radixin-moesin proteins that connect actin filaments to membrane structures. In contrast, NK cell activation by NKG2D induced Vav1 and ezrin-radixin-moesin phosphorylation. Thus, CD94/NKG2A prevents actin-dependent recruitment of raft-associated activation receptors complexes to the activating synapse. This was further substantiated by showing that inhibition of actin polymerization abolished lipid rafts exclusion at the iNKIS, whereas cholesterol depletion had no effect on actin disruption at the iNKIS. These data indicate that the lipid rafts exclusion at the iNKIS is an active process which requires an intact cytoskeleton to maintain lipid rafts outside the inhibitory synapse. The net effect is to maintain an inhibitory state in the proximity of the iNKIS, while allowing the formation of activation synapse at distal points within the same NK cell.

Signalling through NK1.1 triggers NK cells to die but induces NK T cells to produce interleukin-4.

PubMed

Asea, A; Stein-Streilein, J

1998-02-01

In vivo inoculation of specific antibody is an accepted protocol for elimination of specific cell populations. Except for anti-CD3 and anti-CD4, it is not known if the depleted cells are eliminated by signalling through the target molecule or through a more non-specific mechanism. C57BL/6 mice were inoculated with anti-natural killer (NK1.1) monoclonal antibody (mAb). Thereafter spleen cells were harvested, stained for both surface and intracellular markers, and analysed by flow cytometry. As early as 2 hr post inoculation, NK cells were signalled to become apoptotic while signalling through the NK1.1 molecule activated NK1.1+ T-cell receptor (TCR)+ (NK T) cells to increase in number, and produce interleukin-4 (IL-4). Anti NK1.1 mAb was less efficient at signalling apoptosis in NK cells when NK T-cell deficient [beta 2-microglobulin beta 2m-deficient] mice were used compared with wild type mice. Efficient apoptotic signalling was restored when beta 2m-deficient mice were reconstituted with NK T cells. NK-specific antibody best signals the apoptotic process in susceptible NK cells when resistant NK T cells are present, activated, and secrete IL-4.

IL-27 driven upregulation of surface HLA-E expression on monocytes inhibits IFN-γ release by autologous NK cells.

PubMed

Morandi, Fabio; Airoldi, Irma; Pistoia, Vito

2014-01-01

HLA-G and HLA-E are HLA-Ib molecules with several immunoregulatory properties. Their cell surface expression can be modulated by different cytokines. Since IL-27 and IL-30 may either stimulate or regulate immune responses, we have here tested whether these cytokines may modulate HLA-G and -E expression and function on human monocytes. Monocytes expressed gp130 and WSX-1, the two chains of IL27 receptor (R), and IL6Rα (that serves as IL-30R, in combination with gp130). However, only IL27R appeared to be functional, as witnessed by IL-27 driven STAT1/ STAT3 phosphorylation. IL-27, but not IL-30, significantly upregulated HLA-E (but not HLA-G) expression on monocytes. IFN-γ; secretion by activated NK cells was dampened when the latter cells were cocultured with IL-27 pretreated autologous monocytes. Such effect was not achieved using untreated or IL-30 pretreated monocytes, thus indicating that IL-27 driven HLA-E upregulation might be involved, possibly through the interaction of this molecule with CD94/NKG2A inhibitory receptor on NK cells. In contrast, cytotoxic granules release by NK cell in response to K562 cells was unaffected in the presence of IL-27 pretreated monocytes. In conclusion, we delineated a novel immunoregulatory function of IL-27 involving HLA-E upregulation on monocytes that might in turn indirectly impair some NK cell functions.

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. Copyright © 2016 by The American Association of Immunologists, Inc.

Hypoxia Induced Impairment of NK Cell Cytotoxicity against Multiple Myeloma Can Be Overcome by IL-2 Activation of the NK Cells

PubMed Central

Sarkar, Subhashis; Germeraad, Wilfred T. V.; Rouschop, Kasper M. A.; Steeghs, Elisabeth M. P.; van Gelder, Michel; Bos, Gerard M. J.; Wieten, Lotte

2013-01-01

Background Multiple Myeloma (MM) is an incurable plasma cell malignancy residing within the bone marrow (BM). We aim to develop allogeneic Natural Killer (NK) cell immunotherapy for MM. As the BM contains hypoxic regions and the tumor environment can be immunosuppressive, we hypothesized that hypoxia inhibits NK cell anti-MM responses. Methods NK cells were isolated from healthy donors by negative selection and NK cell function and phenotype were examined at oxygen levels representative of hypoxic BM using flowcytometry. Additionally, NK cells were activated with IL-2 to enhance NK cell cytotoxicity under hypoxia. Results Hypoxia reduced NK cell killing of MM cell lines in an oxygen dependent manner. Under hypoxia, NK cells maintained their ability to degranulate in response to target cells, though, the percentage of degranulating NK cells was slightly reduced. Adaptation of NK- or MM cells to hypoxia was not required, hence, the oxygen level during the killing process was critical. Hypoxia did not alter surface expression of NK cell ligands (HLA-ABC, -E, MICA/B and ULBP1-2) and receptors (KIR, NKG2A/C, DNAM-1, NCRs and 2B4). It did, however, decrease expression of the activating NKG2D receptor and of intracellular perforin and granzyme B. Pre-activation of NK cells by IL-2 abrogated the detrimental effects of hypoxia and increased NKG2D expression. This emphasized that activated NK cells can mediate anti-MM effects, even under hypoxic conditions. Conclusions Hypoxia abolishes the killing potential of NK cells against multiple myeloma, which can be restored by IL-2 activation. Our study shows that for the design of NK cell-based immunotherapy it is necessary to study biological interactions between NK- and tumor cells also under hypoxic conditions. PMID:23724099

Impaired plasmacytoid dendritic cell (PDC)-NK cell activity in viremic human immunodeficiency virus infection attributable to impairments in both PDC and NK cell function.

PubMed

Conry, Sara J; Milkovich, Kimberly A; Yonkers, Nicole L; Rodriguez, Benigno; Bernstein, Helene B; Asaad, Robert; Heinzel, Frederick P; Tary-Lehmann, Magdalena; Lederman, Michael M; Anthony, Donald D

2009-11-01

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections impair plasmacytoid dendritic cell (PDC) and natural killer (NK) cell subset numbers and functions, though little is known about PDC-NK cell interactions during these infections. We evaluated PDC-dependent NK cell killing and gamma interferon (IFN-gamma) and granzyme B production, using peripheral blood mononuclear cell (PBMC)-based and purified cell assays of samples from HCV- and HIV-infected subjects. CpG-enhanced PBMC killing and IFN-gamma and granzyme B activity (dependent on PDC and NK cells) were impaired in viremic HIV infection. In purified PDC-NK cell culture experiments, CpG-enhanced, PDC-dependent NK cell activity was cell contact and IFN-alpha dependent, and this activity was impaired in viremic HIV infection but not in HCV infection. In heterologous PDC-NK cell assays, impaired PDC-NK cell killing activity was largely attributable to an NK cell defect, while impaired PDC-NK cell IFN-gamma-producing activity was attributable to both PDC and NK cell defects. Additionally, the response of NK cells to direct IFN-alpha stimulation was defective in viremic HIV infection, and this defect was not attributable to diminished IFN-alpha receptor expression, though IFN-alpha receptor and NKP30 expression was closely associated with killer activity in viremic HIV infection but not in healthy controls. These data indicate that during uncontrolled HIV infection, PDC-dependent NK cell function is impaired, which is in large part attributable to defective IFN-alpha-induced NK cell activity and not to altered IFN-alpha receptor, NKP30, NKP44, NKP46, or NKG2D expression.

Continuously expanding CAR NK-92 cells display selective cytotoxicity against B-cell leukemia and lymphoma.

PubMed

Oelsner, Sarah; Friede, Miriam E; Zhang, Congcong; Wagner, Juliane; Badura, Susanne; Bader, Peter; Ullrich, Evelyn; Ottmann, Oliver G; Klingemann, Hans; Tonn, Torsten; Wels, Winfried S

2017-02-01

Natural killer (NK) cells can rapidly respond to transformed and stressed cells and represent an important effector cell type for adoptive immunotherapy. In addition to donor-derived primary NK cells, continuously expanding cytotoxic cell lines such as NK-92 are being developed for clinical applications. To enhance their therapeutic utility for the treatment of B-cell malignancies, we engineered NK-92 cells by lentiviral gene transfer to express chimeric antigen receptors (CARs) that target CD19 and contain human CD3ζ (CAR 63.z), composite CD28-CD3ζ or CD137-CD3ζ signaling domains (CARs 63.28.z and 63.137.z). Exposure of CD19-positive targets to CAR NK-92 cells resulted in formation of conjugates between NK and cancer cells, NK-cell degranulation and selective cytotoxicity toward established B-cell leukemia and lymphoma cells. Likewise, the CAR NK cells displayed targeted cell killing of primary pre-B-ALL blasts that were resistant to parental NK-92. Although all three CAR NK-92 cell variants were functionally active, NK-92/63.137.z cells were less effective than NK-92/63.z and NK-92/63.28.z in cell killing and cytokine production, pointing to differential effects of the costimulatory CD28 and CD137 domains. In a Raji B-cell lymphoma model in NOD-SCID IL2R γ null mice, treatment with NK-92/63.z cells, but not parental NK-92 cells, inhibited disease progression, indicating that selective cytotoxicity was retained in vivo. Our data demonstrate that it is feasible to generate CAR-engineered NK-92 cells with potent and selective antitumor activity. These cells may become clinically useful as a continuously expandable off-the-shelf cell therapeutic agent. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Tumor suppressive action of indomethacin is NK-cell-independent.

PubMed

Cvetkovska, E; Asea, A; Hellstrand, K; Edström, S

1997-01-01

This study was undertaken to determine whether NK-cells constitute a necessary mediator for the suppression of tumor growth by indomethacin. C57Bl mice with a methylcholantrene (MCG 101) tumor were studied. Indomethacin treatment was provided by daily subcutaneous injections (1 microgram/g body weight). NK-cells were depleted by treatment with a monoclonal antibody to NK1.1. Consecutive indomethacin injections prolonged survival in tumor bearing animals. Indomethacin was equally effective in animals with intact NK-cells as in NK-cell-depleted animals. Further, the MCG cells were apparently insensitive to the lytic activity of NK-cells in vivo. Thus, the clearance of intravenously injected MCG cells from lungs was not affected by depletion of NK-cells in vivo; in contrast, the corresponding clearance of NK-cell-sensitive YAC-1 lymphoma cells was strikingly reduced by the depletion of NK-cells. Our data suggest that NK cells are not a necessary mediator for the suppression of tumor growth by indomethacin.

Liver natural killer cells: subsets and roles in liver immunity

PubMed Central

Peng, Hui; Wisse, Eddie; Tian, Zhigang

2016-01-01

The liver represents a frontline immune organ that is constantly exposed to a variety of gut-derived antigens as a result of its unique location and blood supply. With a predominant role in innate immunity, the liver is enriched with various innate immune cells, among which natural killer (NK) cells play important roles in host defense and in maintaining immune balance. Hepatic NK cells were first described as ‘pit cells' in the rat liver in the 1970s. Recent studies of NK cells in mouse and human livers have shown that two distinct NK cell subsets, liver-resident NK cells and conventional NK (cNK) cells, are present in this organ. Here, we review liver NK cell subsets in different species, revisiting rat hepatic pit cells and highlighting recent progress related to resident NK cells in mouse and human livers, and also discuss the dual roles of NK cells in liver immunity. PMID:26639736

Genetic Causes of Human NK Cell Deficiency and Their Effect on NK Cell Subsets

PubMed Central

Mace, Emily M.; Orange, Jordan S.

2016-01-01

Human NK cells play critical roles in human host defense, particularly the control of viral infection and malignancy, and patients with congenital immunodeficiency affecting NK cell function or number can suffer from severe illness. The importance of NK cell function is particularly underscored in patients with primary immunodeficiency in which NK cells are the primary or sole affected population (NK cell deficiency, NKD). While NKD may lead to the absence of NK cells, we are also gaining an increasing appreciation of the effect that NKD may have on the generation of specific NK cell subsets. In turn, this leads to improved insights into the requirements for human NK cell subset generation, as well as their importance in immune homeostasis. The presence of inherently abnormally developed or functionally impaired NK cells, in particular, appears to be problematic in the way of interfering with normal human host defense and may be more impactful than low numbers of NK cells alone. Here, we review the known genetic causes of NKD and the insight that is derived by these into the requirements for human subset generation and, by extension, for NK cell-mediated immunity. PMID:27994588

Bone marrow-resident NK cells prime monocytes for regulatory function during infection

PubMed Central

Askenase, Michael H.; Han, Seong-Ji; Byrd, Allyson L.; da Fonseca, Denise Morais; Bouladoux, Nicolas; Wilhelm, Christoph; Konkel, Joanne E.; Hand, Timothy W.; Lacerda-Queiroz, Norinne; Su, Xin-Zhuan; Trinchieri, Giorgio; Grainger, John R.; Belkaid, Yasmine

2015-01-01

SUMMARY Tissue-infiltrating Ly6Chi monocytes play diverse roles in immunity, ranging from pathogen killing to immune regulation. How and where this diversity of function is imposed remains poorly understood. Here we show that during acute gastrointestinal infection, priming of monocytes for regulatory function preceded systemic inflammation and was initiated prior to bone marrow egress. Notably, natural killer (NK) cell-derived IFN-γ promoted a regulatory program in monocyte progenitors during development. Early bone marrow NK cell activation was controlled by systemic interleukin-12 (IL-12) produced by Batf3-dependent dendritic cells (DC) in the mucosal-associated lymphoid tissue (MALT). This work challenges the paradigm that monocyte function is dominantly imposed by local signals following tissue recruitment, and instead proposes a sequential model of differentiation in which monocytes are pre-emptively educated during development in the bone marrow to promote their tissue-specific function. PMID:26070484

Classification of human natural killer cells based on migration behavior and cytotoxic response.

PubMed

Vanherberghen, Bruno; Olofsson, Per E; Forslund, Elin; Sternberg-Simon, Michal; Khorshidi, Mohammad Ali; Pacouret, Simon; Guldevall, Karolin; Enqvist, Monika; Malmberg, Karl-Johan; Mehr, Ramit; Önfelt, Björn

2013-02-21

Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas a minority of NK cells was responsible for a majority of the target cell deaths. These dynamic cytotoxicity data allowed categorization of NK cells into 5 distinct classes. A small but particularly active subclass of NK cells killed several target cells in a consecutive fashion. These "serial killers" delivered their lytic hits faster and induced faster target cell death than other NK cells. Fast, necrotic target cell death was correlated with the amount of perforin released by the NK cells. Our data are consistent with a model in which a small fraction of NK cells drives tumor elimination and inflammation.

Bone marrow produces sufficient alloreactive natural killer (NK) cells in vivo to cure mice from subcutaneously and intravascularly injected 4T1 breast cancer.

PubMed

van Gelder, Michel; Vanclée, Ariane; van Elssen, Catharina H M J; Hupperets, Pierre; Wieten, Lotte; Bos, Gerard M

2017-02-01

Administration of 5 million alloreactive natural killer (NK) cells after low-dose chemo-irradiation cured mice of 4T1 breast cancer, supposedly dose dependent. We now explored the efficacy of bone marrow as alternative in vivo source of NK cells for anti-breast cancer treatment, as methods for in vitro clinical scale NK cell expansion are still in developmental phases. Progression-free survival (PFS) after treatment with different doses of spleen-derived alloreactive NK cells to 4T1-bearing Balb/c mice was measured to determine a dose-response relation. The potential of bone marrow as source of alloreactive NK cells was explored using MHC-mismatched mice as recipients of 4T1. Chemo-irradiation consisted of 2× 2 Gy total body irradiation and 200 mg/kg cyclophosphamide. Antibody-mediated in vivo NK cell depletion was applied to demonstrate the NK cell's role. Administration of 2.5 instead of 5 million alloreactive NK cells significantly reduced PFS, evidencing dose responsiveness. Compared to MHC-matched receivers of subcutaneous 4T1, fewer MHC-mismatched mice developed tumors, which was due to NK cell alloreactivity because in vivo NK cell depletion facilitated tumor growth. Application of low-dose chemo-irradiation increased plasma levels of NK cell-activating cytokines, NK cell activity and enhanced NK cell-dependent elimination of subcutaneous tumors. Intravenously injected 4T1 was eliminated by alloreactive NK cells in MHC-mismatched recipients without the need for chemo-irradiation. Bone marrow is a suitable source of sufficient alloreactive NK cells for the cure of 4T1 breast cancer. These results prompt clinical exploration of bone marrow transplantation from NK-alloreactive MHC-mismatched donors in patients with metastasized breast cancer.

Antibody-dependent cellular cytotoxicity and cytokine/chemokine secretion by KHYG-1 cells stably expressing FcγRIIIA.

PubMed

Kobayashi, Eiji; Motoi, Sotaro; Sugiura, Masahito; Kajikawa, Masunori; Kojima, Shuji; Kohroki, Junya; Masuho, Yasuhiko

2014-09-01

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC. Copyright © 2014 Elsevier B.V. All rights reserved.

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

PubMed

Paust, Silke; Gill, Harvinder S; Wang, Bao-Zhong; Flynn, Michael P; Moseman, E Ashley; Senman, Balimkiz; Szczepanik, Marian; Telenti, Amalio; Askenase, Philip W; Compans, Richard W; von Andrian, Ulrich H

2010-12-01

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

N6-isopentenyladenosine, an endogenous isoprenoid end product, directly affects cytotoxic and regulatory functions of human NK cells through FDPS modulation.

PubMed

Ciaglia, Elena; Pisanti, Simona; Picardi, Paola; Laezza, Chiara; Malfitano, Anna Maria; D'Alessandro, Alba; Gazzerro, Patrizia; Vitale, Mario; Carbone, Ennio; Bifulco, Maurizio

2013-12-01

iPA is a naturally occurring nucleoside with an isopentenyl moiety derived from the mevalonate pathway and a well-established anti-tumor activity. In analogy to the unique specificity for phosphoantigens, such as IPP, shown by human Vγ9Vδ2 T cells, here, we report for the first time the ability of iPA to selectively expand and directly target human NK cells. Interestingly, submicromolar doses of iPA stimulate resting human NK cells and synergize with IL-2 to induce a robust activation ex vivo with significant secretion of CCL5 and CCL3 and a large increase in TNF-α and IFN-γ production when compared with IL-2 single cytokine treatment. Moreover, iPA promotes NK cell proliferation and up-regulates the expression of specific NK cell-activating receptors, as well as CD69 and CD107a expression. Accordingly, this phenotype correlates with significantly greater cytotoxicity against tumor targets. At the molecular level, iPA leads to a selective, potent activation of MAPK signaling intermediaries downstream of the IL-2R. The effect results, at least in part, from the fine modulation of the FDPS activity, the same enzyme implicated in the stimulation of the human γδ T cells. The iPA-driven modulation of FDPS can cause an enhancement of post-translational prenylation essential for the biological activity of key proteins in NK signaling and effector functions, such as Ras. These unanticipated properties of iPA provide an additional piece of evidence of the immunoregulatory role of the intermediates of the mevalonate pathway and open novel therapeutic perspectives for this molecule as an immune-modulatory drug.

Viral Evasion of Natural Killer Cell Activation

PubMed Central

Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

2016-01-01

Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections. PMID:27077876

Viral Evasion of Natural Killer Cell Activation.

PubMed

Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

2016-04-12

Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections.

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

NK-1 receptor desensitization and neutral endopeptidase terminate SP-induced pancreatic plasma extravasation.

PubMed

Maa, J; Grady, E F; Kim, E H; Yoshimi, S K; Hutter, M M; Bunnett, N W; Kirkwood, K S

2000-10-01

Substance P (SP) induces plasma extravasation and neutrophil infiltration by activating the neurokinin-1 receptor (NK1-R). We characterized the mechanisms regulating this response in the rat pancreas. Anesthetized rats were continuously infused with SP, and plasma extravasation was quantified using Evans blue (EB) dye. Continuous infusion of SP (8 nmol. kg(-1). h(-1)) resulted in a threshold increase in EB at 15 min, a peak effect at 30 min (150% increase), and a return to baseline by 60 min. The NK1-R antagonist CP-96,345 blocked SP-induced plasma extravasation. After 60 min, the NK1-R was desensitized to agonist challenge. Resensitization was first detected at 20 min and increased until full recovery was seen at 30 min. Inhibition of the cell-surface protease neutral endopeptidase (NEP) by phosphoramidon potentiated the effect of exogenous SP; therefore endogenous NEP attenuates SP-induced plasma extravasation. Thus the continuous infusion of SP stimulates plasma extravasation in the rat pancreas via activation of the NK1-R, and these effects are terminated by both desensitization of the NK1-R and the cell-surface protease NEP.

Suppression of innate immunity (natural killer cell/interferon-γ) in the advanced stages of liver fibrosis in mice

PubMed Central

Jeong, Won-Il; Park, Ogyi; Suh, Yang-Gun; Byun, Jin-Seok; Park, So-Young; Choi, Earl; Kim, Ja-Kyung; Ko, Hyojin; Wang, Hua; Miller, Andrew M.; Gao, Bin

2011-01-01

Activation of innate immunity (natural killer cell/interferon-γ: NK cell/IFN-γ) has been shown to play an important role in anti-viral and anti-tumor defenses as well as anti-fibrogenesis. However, little is known about the regulation of innate immunity during chronic liver injury. Here, we compared the functions of NK cells in early and advanced liver fibrosis induced by a 2-week or a 10 week-carbon tetrachloride (CCl4) challenge, respectively. Injection of poly I:C or IFN-γ induced NK cell activation and NK cell killing of hepatic stellate cells (HSCs) in the 2-week CCl4 model. Such activation was diminished in the 10-week CCl4 model. Consistent with these findings, the inhibitory effect of poly I:C and IFN-γ on liver fibrosis was markedly reduced in the 10-week vs. the 2-week CCl4 model. In vitro co-culture experiments demonstrated that 4-day cultured (early-activated) HSCs induce NK cell activation via an NKG2D-retinoic acid-induced early gene 1 (RAE1)-dependent mechanism. Such activation was reduced when co-cultured with 8-day cultured (intermediately-activated) HSCs due to the production of transforming growth factor-β (TGF-β) by HSCs. Moreover, early-activated HSCs were sensitive, while intermediately-activated HSCs were resistant to IFN-γ mediated inhibition of cell proliferation, likely due to elevated expression of suppressor of cytokine signaling 1 (SOCS1). Disruption of the SOCS1 gene restored the IFN-γ inhibition of cell proliferation in intermediately-activated HSCs. Production of retinol metabolites by HSCs contributed to SOCS1 induction and subsequently inhibited IFN-γ signaling and functioning, while production of TGF-β by HSCs inhibited NK cell function and cytotoxicity against HSCs. Conclusion The anti-fibrogenic effects of NK cell/IFN-γ are suppressed during advanced liver injury, which is likely due to the increased production of TGF-β and expression of SOCS1 in intermediately-activated HSCs. PMID:21480338

Tricking the balance: NK cells in anti-cancer immunity.

PubMed

Pahl, Jens; Cerwenka, Adelheid

2017-01-01

Natural Killer (NK) cells are classically considered innate immune effector cells involved in the first line of defense against infected and malignant cells. More recently, NK cells have emerged to acquire properties of adaptive immunity in response to certain viral infections such as expansion of specific NK cell subsets and long-lasting virus-specific responses to secondary challenges. NK cells distinguish healthy cells from abnormal cells by measuring the net input of activating and inhibitory signals perceived from target cells through NK cell surface receptors. Acquisition of activating ligands in combination with reduced expression of MHC class I molecules on virus-infected and cancer cells activates NK cell cytotoxicity and release of immunostimulatory cytokines like IFN-γ. In the cancer microenvironment however, NK cells become functionally impaired by inhibitory factors produced by immunosuppressive immune cells and cancer cells. Here we review recent progress on the role of NK cells in cancer immunity. We describe regulatory factors of the tumor microenvironment on NK cell function which determine cancer cell destruction or escape from immune recognition. Finally, recent strategies that focus on exploiting NK cell anti-cancer responses for immunotherapeutic approaches are outlined. Copyright © 2015 Elsevier GmbH. All rights reserved.

NK Cell-derived Exosomes From NK Cells Previously Exposed to Neuroblastoma Cells Augment the Antitumor Activity of Cytokine-activated NK Cells.

PubMed

Shoae-Hassani, Alireza; Hamidieh, Amir Ali; Behfar, Maryam; Mohseni, Rashin; Mortazavi-Tabatabaei, Seyed A; Asgharzadeh, Shahab

2017-09-01

Immune cell-derived exosomes can increase immunity against tumors. In contrast, tumor-derived exosomes can reduce the immunity and can change the tumor microenvironment to further develop and provide metastasis. These effects take place by an alteration in the innate and adaptive immune cell functions. In this experiment, we studied the natural killer (NK) cells' effectiveness on tumor cells after expansion and thereafter incubated it with exosomes. The exosomes were derived from 2 populations of NK cells: (1) naive NK cells and, (2) NK cells previously exposed to neuroblastoma (NB) cells. Moreover, we have studied the NB-derived exosomes on NK cell function. The molecular load of the characterized exosomes (by means of nanoparticle-tracking analysis, flow cytometry, scanning electron microscopy, and western blot) from NK cells exposed to the NB cell revealed their expression of natural killer cell receptors in addition to CD56, NKG2D, and KIR2DL2 receptors. These exosomes were used to treat NK cells and thereafter administered to NB tumor cells both in vitro and in vivo. Our results showed some kind of NK cells' education by the exosomes. This education from NK cells previously exposed to NB cell-derived exosomes caused efficient and greater cytotoxicity against NB tumors, but NB-derived exosomes act as tumor promoters by providing a tumor supporting niche. Hence, this method of preparing the exosomes has a dramatic effect on activation of anti-NK cells against NB cells.

Natural Killer Dendritic Cells Enhance Immune Responses Elicited by α -Galactosylceramide-Stimulated Natural Killer T Cells.

PubMed

Lee, Sung Won; Park, Hyun Jung; Kim, Nayoung; Hong, Seokmann

2013-01-01

Natural killer dendritic cells (NKDCs) possess potent anti-tumor activity, but the cellular effect of NKDC interactions with other innate immune cells is unclear. In this study, we demonstrate that the interaction of NKDCs and natural killer T (NKT) cells is required for the anti-tumor immune responses that are elicited by α -galactosylceramide ( α -GC) in mice. The rapid and strong expression of interferon- γ by NKDCs after α -GC stimulation was dependent on NKT cells. Various NK and DC molecular markers and cytotoxic molecules were up-regulated following α -GC administration. This up-regulation could improve NKDC presentation of tumor antigens and increase cytotoxicity against tumor cells. NKDCs were required for the stimulation of DCs, NK cells, and NKT cells. The strong anti-tumor immune responses elicited by α -GC may be due to the down-regulation of regulatory T cells. Furthermore, the depletion of NKDCs dampened the tumor clearance mediated by α -GC-stimulated NKT cells in vivo. Taken together, these results indicate that complex interactions of innate immune cells might be required to achieve optimal anti-tumor immune responses during the early stages of tumorigenesis.

Low baseline levels of NK cells may predict a positive response to ipilimumab in melanoma therapy.

PubMed

Tietze, Julia K; Angelova, Daniela; Heppt, Markus V; Ruzicka, Thomas; Berking, Carola

2017-07-01

The introduction of immune checkpoint blockade (ICB) has been a breakthrough in the therapy of metastatic melanoma. The influence of ICB on T-cell populations has been studied extensively, but little is known about the effect on NK cells. In this study, we analysed the relative and absolute amounts of NK cells and of the subpopulations of CD56 dim and CD56 bright NK cells among the peripheral blood mononuclear cells (PBMCs) of 32 patients with metastatic melanoma before and under treatment with ipilimumab or pembrolizumab by flow cytometry. In 15 (47%) patients, an abnormal low amount of NK cells was found at baseline. Analysis of the subpopulations showed also low or normal baseline levels for CD56 dim NK cells, whereas the baseline levels of CD56 bright NK cells were either normal or abnormally high. The relative and absolute amounts of NK cells and of CD56 dim and CD56 bright NK cell subpopulations in patients with a normal baseline did not change under treatment. However, patients with a low baseline of NK cells and CD56 dim NK cells showed a significant increase in these immune cell subsets, but the amounts remained to be lower than the normal baseline. The amount of CD56 bright NK cells was unaffected by treatment. The baseline levels of NK cells were correlated with the number of metastatic organs. Their proportion increased, whereas the expression of NKG2D decreased significantly when more than one organ was affected by metastases. Low baseline levels of NK cells and CD56 dim NK cells as well as normal baseline levels of CD56 bright NK cells correlated significantly with a positive response to ipilimumab but not to pembrolizumab. Survival curves of patients with low amounts of CD56 dim NK cells treated with ipilimumab showed a trend to longer survival. Normal baseline levels of CD56 bright NK cells were significantly correlated with longer survival as compared to patients with high baseline levels. In conclusion, analysis of the amounts of total NK cells and of CD56 dim and CD56 bright NK cells subpopulations at baseline may help to predict the outcome of treatment with ipilimumab. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Astrocyte-derived interleukin-15 exacerbates ischemic brain injury via propagation of cellular immunity.

PubMed

Li, Minshu; Li, Zhiguo; Yao, Yang; Jin, Wei-Na; Wood, Kristofer; Liu, Qiang; Shi, Fu-Dong; Hao, Junwei

2017-01-17

Astrocytes are believed to bridge interactions between infiltrating lymphocytes and neurons during brain ischemia, but the mechanisms for this action are poorly understood. Here we found that interleukin-15 (IL-15) is dramatically up-regulated in astrocytes of postmortem brain tissues from patients with ischemic stroke and in a mouse model of transient focal brain ischemia. We generated a glial fibrillary acidic protein (GFAP) promoter-controlled IL-15-expressing transgenic mouse (GFAP-IL-15 tg ) line and found enlarged brain infarcts, exacerbated neurodeficits after the induction of brain ischemia. In addition, knockdown of IL-15 in astrocytes attenuated ischemic brain injury. Interestingly, the accumulation of CD8 + T and natural killer (NK) cells was augmented in these GFAP-IL-15 tg mice after brain ischemia. Of note, depletion of CD8 + T or NK cells attenuated ischemic brain injury in GFAP-IL-15 tg mice. Furthermore, knockdown of the IL-15 receptor α or blockade of cell-to-cell contact diminished the activation and effector function of CD8 + T and NK cells in GFAP-IL-15 tg mice, suggesting that astrocytic IL-15 is delivered in trans to target cells. Collectively, these findings indicate that astrocytic IL-15 could aggravate postischemic brain damage via propagation of CD8 + T and NK cell-mediated immunity.

Irradiated and activated autologous PBMCs induce expansion of highly cytotoxic human NK cells in vitro.

PubMed

Ahn, Yong-Oon; Kim, Saerom; Kim, Tae Min; Song, Eun Young; Park, Myoung Hee; Heo, Dae Seog

2013-09-01

Adoptive cell transfer of ex vivo-activated natural killer (NK) cells is a promising therapy for cancer treatment. Because of inhibitory signaling through killer immunoglobulin-like receptor (KIR)-KIR ligands, KIR-mismatched allogeneic NK cell transfer is considered to be a more effective strategy than is autologous transfer. However, purified NK cells do not expand well enough in vitro with good manufacturing practice-compliant components for clinical use. Some investigators have developed selective expansion of NK cells from peripheral blood mononuclear cells, but these cells have the risk of graft-versus-host disease in allogeneic settings because of T cells contamination. In this study, we developed a novel method for NK cell activation and expansion. Using only good manufacturing practice-compliant components and autologous feeder cells, once purified NK cells were effectively expanded (2500-fold at day 17). The expanded cells were highly purified NK cells, and the use of these cells is suitable for allogeneic transfer without the risk of graft-versus-host disease induction. Importantly, the expanded NK cells also showed enhanced cytotoxicity compared with NK cells conventionally expanded by recombinant human interleukin 2. Finally, induction of NKG2D ligand expression on feeder cells implies that the NKG2D-NKG2DL interaction may play a role in NK cell expansion. In conclusion, this method can be used to obtain NK cells for more successful allogeneic NK cell adoptive transfer for use in antitumor immune therapy.

Adoptive transfer of natural killer cells promotes the anti-tumor efficacy of T cells.

PubMed

Goding, Stephen R; Yu, Shaohong; Bailey, Lisa M; Lotze, Michael T; Basse, Per H

2017-04-01

The density of NK cells in tumors correlates positively with prognosis in many types of cancers. The average number of infiltrating NK cells is, however, quite modest (approximately 30 NK cells/sq.mm), even in tumors deemed to have a "high" density of infiltrating NK cells. It is unclear how such low numbers of tumor-infiltrating NK cells can influence outcome. Here, we used ovalbumin-expressing tumor cell lines and TCR transgenic, OVA-specific cytotoxic T lymphocytes (OT-I-CTLs) to determine whether the simultaneous attack by anti-tumor CTLs and IL-2-activated NK (A-NK) cells synergistically increases the overall tumor cell kill and whether upregulation of tumor MHC class-I by NK cell-derived interferon-gamma (IFNγ) improves tumor-recognition and kill by anti-tumor CTLs. At equal E:T ratios, A-NK cells killed OVA-expressing tumor cells better than OT-I-CTLs. The cytotoxicity against OVA-expressing tumor cells increased by combining OT-I-CTLs and A-NK cells, but the increase was additive rather than synergistic. A-NK cells adenovirally-transduced to produce IL-12 (A-NK IL-12 ) produced high amounts of IFNγ. The addition of a low number of A-NK IL-12 cells to OT-I-CTLs resulted in a synergistic, albeit modest, increase in overall cytotoxicity. Pre-treatment of tumor cells with NK cell-conditioned medium increased tumor MHC expression and sensitivity to CTL-mediated killing. Pre-treatment of CTLs with NK cell-conditioned medium had no effect on CTL cytotoxicity. In vivo, MHC class-I expression by OVA-expressing B16 melanoma lung metastases increased significantly within 24-48h after adoptive transfer of A-NK IL-12 cells. OT-I-CTLs and A-NK IL-12 cells localized selectively and equally well into OVA-expressing B16 lung metastases and treatment of mice bearing 7-days-old OVA-B16 lung metastases with both A-NK IL-12 cells and OT-I-CTLs lead to a significant prolongation of survival. Thus, an important function of tumor-infiltrating NK cells may be to increase tumor cell expression of MHC class-I through secretion of IFNγ, to prepare them for recognition by tumor-specific CTLs. Copyright © 2016 Elsevier Inc. All rights reserved.

Cognate HLA absence in trans diminishes human NK cell education

PubMed Central

Landtwing, Vanessa; Raykova, Ana; Pezzino, Gaetana; Béziat, Vivien; Graf, Claudine; Moretta, Alessandro; Capaul, Riccarda; Zbinden, Andrea; Malmberg, Karl-Johan; Chijioke, Obinna; Münz, Christian

2016-01-01

NK cells are innate lymphocytes with protective functions against viral infections and tumor formation. Human NK cells carry inhibitory killer cell Ig-like receptors (KIRs), which recognize distinct HLAs. NK cells with KIRs for self-HLA molecules acquire superior cytotoxicity against HLA– tumor cells during education for improved missing-self recognition. Here, we reconstituted mice with human hematopoietic cells from donors with homozygous KIR ligands or with a mix of hematopoietic cells from these homozygous donors, allowing assessment of the resulting KIR repertoire and NK cell education. We found that co-reconstitution with 2 KIR ligand–mismatched compartments did not alter the frequency of KIR-expressing NK cells. However, NK cell education was diminished in mice reconstituted with parallel HLA compartments due to a lack of cognate HLA molecules on leukocytes for the corresponding KIRs. This change in NK cell education in mixed human donor–reconstituted mice improved NK cell–mediated immune control of EBV infection, indicating that mixed hematopoietic cell populations could be exploited to improve NK cell reactivity against leukotropic pathogens. Taken together, these findings indicate that leukocytes lacking cognate HLA ligands can disarm KIR+ NK cells in a manner that may decrease HLA– tumor cell recognition but allows for improved NK cell–mediated immune control of a human γ-herpesvirus. PMID:27571408

Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor.

PubMed

Schmidlin, F; Dery, O; DeFea, K O; Slice, L; Patierno, S; Sternini, C; Grady, E F; Bunnett, N W

2001-07-06

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.

CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets

PubMed Central

Mao, Hsiaoyin C.; Wei, Min; Hughes, Tiffany; Zhang, Jianying; Park, Il-kyoo; Liu, Shujun; McClory, Susan; Marcucci, Guido; Trotta, Rossana

2010-01-01

Human CD56bright natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs), high interferon-γ (IFN-γ) production, but little cytotoxicity. CD56dim NK cells have high KIR expression, produce little IFN-γ, yet display high cytotoxicity. We hypothesized that, if human NK maturation progresses from a CD56bright to a CD56dim phenotype, an intermediary NK cell must exist, which demonstrates more functional overlap than these 2 subsets, and we used CD94 expression to test our hypothesis. CD94highCD56dim NK cells express CD62L, CD2, and KIR at levels between CD56bright and CD94lowCD56dim NK cells. CD94highCD56dim NK cells produce less monokine-induced IFN-γ than CD56bright NK cells but much more than CD94lowCD56dim NK cells because of differential interleukin-12–mediated STAT4 phosphorylation. CD94highCD56dim NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56bright NK cells but lower than CD94lowCD56dim NK cells. Collectively, our data suggest that the density of CD94 surface expression on CD56dim NK cells identifies a functional and likely developmental intermediary between CD56bright and CD94lowCD56dim NK cells. This supports the notion that, in vivo, human CD56bright NK cells progress through a continuum of differentiation that ends with a CD94lowCD56dim phenotype. PMID:19897577

CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets.

PubMed

Yu, Jianhua; Mao, Hsiaoyin C; Wei, Min; Hughes, Tiffany; Zhang, Jianying; Park, Il-kyoo; Liu, Shujun; McClory, Susan; Marcucci, Guido; Trotta, Rossana; Caligiuri, Michael A

2010-01-14

Human CD56(bright) natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs), high interferon-gamma (IFN-gamma) production, but little cytotoxicity. CD56(dim) NK cells have high KIR expression, produce little IFN-gamma, yet display high cytotoxicity. We hypothesized that, if human NK maturation progresses from a CD56(bright) to a CD56(dim) phenotype, an intermediary NK cell must exist, which demonstrates more functional overlap than these 2 subsets, and we used CD94 expression to test our hypothesis. CD94(high)CD56(dim) NK cells express CD62L, CD2, and KIR at levels between CD56(bright) and CD94(low)CD56(dim) NK cells. CD94(high)CD56(dim) NK cells produce less monokine-induced IFN-gamma than CD56(bright) NK cells but much more than CD94(low)CD56(dim) NK cells because of differential interleukin-12-mediated STAT4 phosphorylation. CD94(high)CD56(dim) NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56(bright) NK cells but lower than CD94(low)CD56(dim) NK cells. Collectively, our data suggest that the density of CD94 surface expression on CD56(dim) NK cells identifies a functional and likely developmental intermediary between CD56(bright) and CD94(low)CD56(dim) NK cells. This supports the notion that, in vivo, human CD56(bright) NK cells progress through a continuum of differentiation that ends with a CD94(low)CD56(dim) phenotype.

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

PubMed Central

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

2015-01-01

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. PMID:26073325

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

Queirós, Maria Luís; Gonçalves, Marta; Fonseca, Sónia; Moura, João

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

IL15 Infusion of Cancer Patients Expands the Subpopulation of Cytotoxic CD56bright NK Cells and Increases NK-Cell Cytokine Release Capabilities.

PubMed

Dubois, Sigrid; Conlon, Kevin C; Müller, Jürgen R; Hsu-Albert, Jennifer; Beltran, Nancy; Bryant, Bonita R; Waldmann, Thomas A

2017-10-01

The cytokine IL15 is required for survival and activation of natural killer (NK) cells as well as expansion of NK-cell populations. Here, we compare the effects of continuous IL15 infusions on NK-cell subpopulations in cancer patients. Infusions affected the CD56 bright NK-cell subpopulation in that the expansion rates exceeded those of CD56 dim NK-cell populations with a 350-fold increase in their total cell numbers compared with 20-fold expansion for the CD56 dim subset. CD56 bright NK cells responded with increased cytokine release to various stimuli, as expected given their immunoregulatory functions. Moreover, CD56 bright NK cells gained the ability to kill various target cells at levels that are typical for CD56 dim NK cells. Some increased cytotoxic activities were also observed for CD56 dim NK cells. IL15 infusions induced expression changes on the surface of both NK-cell subsets, resulting in a previously undescribed and similar phenotype. These data suggest that IL15 infusions expand and arm CD56 bright NK cells that alone or in combination with tumor-targeting antibodies may be useful in the treatment of cancer. Cancer Immunol Res; 5(10); 929-38. ©2017 AACR . ©2017 American Association for Cancer Research.

Copy Number Variation of KIR Genes Influences HIV-1 Control

PubMed Central

Shianna, Kevin V.; Feng, Sheng; Urban, Thomas J.; Ge, Dongliang; De Luca, Andrea; Martinez-Picado, Javier; Wolinsky, Steven M.; Martinson, Jeremy J.; Jamieson, Beth D.; Bream, Jay H.; Martin, Maureen P.; Borrow, Persephone; Letvin, Norman L.; McMichael, Andrew J.; Haynes, Barton F.; Telenti, Amalio; Carrington, Mary; Goldstein, David B.; Alter, Galit

2011-01-01

A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection. PMID:22140359

NKp44 expression, phylogenesis and function in non-human primate NK cells

PubMed Central

De Maria, Andrea; Ugolotti, Elisabetta; Rutjens, Erik; Mazza, Stefania; Radic, Luana; Faravelli, Alessandro; Koopman, Gerrit; Di Marco, Eddi; Costa, Paola; Ensoli, Barbara; Cafaro, Aurelio; Mingari, Maria Cristina; Moretta, Lorenzo; Heeney, Jonathan

2009-01-01

Molecular and functional characterization of the natural cytotoxicity receptor (NCR) NKp44 in species other than Homo sapiens has been elusive, so far. Here, we provide complete phenotypic, molecular and functional characterization for NKp44 triggering receptor on Pan troglodytes NK cells, the closest human relative, and the analysis of NKp44-genomic locus and transcription in Macaca fascicularis. Similar to H. sapiens, NKp44 expression is detectable on chimpanzee NK cells only upon activation. However, basal NKp44 transcription is 5-fold higher in chimpanzees with lower differential increases upon cell activation compared with humans. Upon activation, an overall 12-fold lower NKp44 gene expression is observed in P. troglodytes compared with H. sapiens NK cells with only a slight reduction in NKp44 surface expression. Functional analysis of ‘in vitro’ activated purified NK cells confirms the NKp44 triggering potential compared with other major NCRs. These findings suggest the presence of a post-transcriptional regulation that evolved differently in H. sapiens. Analysis of cynomolgus NKp44-genomic sequence and transcription pattern showed very low levels of transcription with occurrence of out-of-frame transcripts and no surface expression. The present comparative analysis suggests that NKp44-genomic organization appears during macaque speciation, with considerable evolution of its transcriptional and post-transcriptional tuning. Thus, NKp44 may represent an NCR being only recently emerged during speciation, acquiring functional relevance only in non-human primates closest to H. sapiens. PMID:19147838

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

PubMed

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

2016-09-01

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.

High expression of NKG2A/CD94 and low expression of granzyme B are associated with reduced cord blood NK cell activity.

PubMed

Wang, Yanyan; Xu, Han; Zheng, Xiaodong; Wei, Haiming; Sun, Rui; Tian, Zhigang

2007-10-01

Human umbilical cord blood (CB) has recently been used as a source of stem cells in transplantation. NK cells derived from CB are the key effector cells involved in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL). It was reported that the activity of CB NK cells was lower than that of adult peripheral blood (PB) NK cells. In this study, we analyzed the expression of some NK cell receptors and cytotoxicity-related molecules in CB and PB NK cells. The expressions of activating NK receptors, CD16, NKG2D and NKp46, did not show significant difference between CB and PB NK cells. But the expression of inhibitory receptor NKG2A/CD94 was significantly higher on CB NK cells. As to the effector function molecules, granzyme B was expressed significantly lower in CB NK cells, but the expressions of intracellular perforin, IFN-gamma, TNF-alpha and cell surface FasL and TRAIL did not show difference between CB and PB NK cells. The results indicated that the high expression of NKG2A/CD94 and low expression of granzyme B may be related with the reduced activity of CB NK cells.

CD16A Activation of NK Cells Promotes NK Cell Proliferation and Memory-Like Cytotoxicity against Cancer Cells.

PubMed

Pahl, Jens H W; Koch, Joachim; Götz, Jana-Julia; Arnold, Annette; Reusch, Uwe; Gantke, Thorsten; Rajkovic, Erich; Treder, Martin; Cerwenka, Adelheid

2018-05-01

CD16A is a potent cytotoxicity receptor on human natural killer (NK) cells, which can be exploited by therapeutic bispecific antibodies. So far, the effects of CD16A-mediated activation on NK cell effector functions beyond classical antibody-dependent cytotoxicity have remained poorly elucidated. Here, we investigated NK cell responses after exposure to therapeutic antibodies such as the tetravalent bispecific antibody AFM13 (CD30/CD16A), designed for the treatment of Hodgkin lymphoma and other CD30 + lymphomas. Our results reveal that CD16A engagement enhanced subsequent IL2- and IL15-driven NK cell proliferation and expansion. This effect involved the upregulation of CD25 (IL2Rα) and CD132 (γ c ) on NK cells, resulting in increased sensitivity to low-dose IL2 or to IL15. CD16A engagement initially induced NK cell cytotoxicity. The lower NK cell reactivity observed 1 day after CD16A engagement could be recovered by reculture in IL2 or IL15. After reculture in IL2 or IL15, these CD16A-experienced NK cells exerted more vigorous IFNγ production upon restimulation with tumor cells or cytokines. Importantly, after reculture, CD16A-experienced NK cells also exerted increased cytotoxicity toward different tumor targets, mainly through the activating NK cell receptor NKG2D. Our findings uncover a role for CD16A engagement in priming NK cell responses to restimulation by cytokines and tumor cells, indicative of a memory-like functionality. Our study suggests that combination of AFM13 with IL2 or IL15 may boost NK cell antitumor activity in patients by expanding tumor-reactive NK cells and enhancing NK cell reactivity, even upon repeated tumor encounters. Cancer Immunol Res; 6(5); 517-27. ©2018 AACR . ©2018 American Association for Cancer Research.

Malignant hematopoietic cell lines: in vitro models for the study of natural killer cell leukemia-lymphoma.

PubMed

Drexler, H G; Matsuo, Y

2000-05-01

Malignancies involving natural killer (NK) cells are rare disorders. The complexity of NK cell-involving disorders has only recently been appreciated. Modern classifications discern immature (precursor) from mature NK cell leukemias-lymphomas. Continuous NK leukemia-lymphoma cell lines represent important model systems to study these neoplasms. While there are a number of putative NK cell lines which are, however, either not characterized, not immortalized, non-malignant, non-NK, or plain false cell lines, six bona fide malignant NK cell lines have been established and are sufficiently well characterized: HANK1, KHYG-1, NK-92, NKL, NK-YS and YT. Except for YT which was derived from a not further defined acute lymphoblastic lymphoma, these cell lines were established from patients with various NK cell malignancies. Five of the six cell lines are constitutively interleukin-2-dependent. Their immunoprofile is remarkably similar: CD1-, CD2+, surface CD3 (but cytoplasmic CD3epsilon+), CD4-, CD5-, CD7+, CD8-, CD16-, CD56+, CD57-, TCRalphabeta-, TCRgammadelta-, negative for B cell and myelomonocytic markers. The immunoglobulin heavy chain and T cell receptor genes are all in germline configuration. All six lines show complex chromosomal alterations, with both numerical and structural aberrations, attesting to their malignant and monoclonal nature. Functionally, these cells which contain azurophilic granules in their cytoplasm are nearly universally positive in NK activity assays. Three of five cell lines are Epstein-Barr virus-positive (type II latency). The composite data on these six cell lines allow for the operational definition of a typical malignant NK cell line profile. NK leukemia-lymphoma cell lines will prove invaluable for studies of normal and malignant NK cell biology.

Effect of IL-18 on the Expansion and Phenotype of Human Natural Killer Cells: Application to Cancer Immunotherapy.

PubMed

Senju, Hiroaki; Kumagai, Asuka; Nakamura, Yoichi; Yamaguchi, Hiroyuki; Nakatomi, Katsumi; Fukami, Shota; Shiraishi, Kengo; Harada, Yuka; Nakamura, Mitsuhiro; Okamura, Haruki; Tanaka, Yoshimasa; Mukae, Hiroshi

2018-01-01

When pathogenic stresses are recognized by innate immune cells, inflammasomes are assembled and caspase-1 is activated, resulting in the conversion of pro-IL-18 into mature IL-18. Because natural killer (NK) cells express IL-18 receptors, IL-18 may play roles in immune functions of NK cells. In the present study, we examined the effect of IL-18 on NK cells derived from lung cancer patients and healthy adult volunteers. When peripheral blood NK cells were stimulated with IL-2, the cells formed clusters beginning on day 5-6 and proliferated thereafter, in which the number of NK cells increased by 10-fold in 10 days. When IL-18 was added, cell clusters were observed as early as on day 4 and NK cells proliferated vigorously. On day 10, the expansion rate was 56-fold on average, showing that IL-18 promoted the expansion of NK cells. It was also notable that IL-18 enhanced the expression of CD80, CD86, HLA-DR and HLA-DQ on NK cells, suggesting that IL-18 conferred NK cells an APC-like phenotype. When cellular cytotoxicity was determined, APC-like NK cells efficiently killed tumor cells and anti-tumor activity was augmented by the addition of tumor antigen-specific mAbs. In addition, IFN-γ was produced by APC-like NK cells in response to tumor cells, and the cytokine production was further enhanced by mAbs. Taken together, IL-18 not only promoted the expansion of NK cells, but also changed the phenotype of NK cells. IL-2/IL-18-induced NK cells might, therefore, serve as a bridge between innate immunity and adaptive immunity and be useful for cancer immunotherapy.

Natural Killer Cell-Based Therapies Targeting Cancer: Possible Strategies to Gain and Sustain Anti-Tumor Activity

PubMed Central

Dahlberg, Carin I. M.; Sarhan, Dhifaf; Chrobok, Michael; Duru, Adil D.; Alici, Evren

2015-01-01

Natural killer (NK) cells were discovered 40 years ago, by their ability to recognize and kill tumor cells without the requirement of prior antigen exposure. Since then, NK cells have been seen as promising agents for cell-based cancer therapies. However, NK cells represent only a minor fraction of the human lymphocyte population. Their skewed phenotype and impaired functionality during cancer progression necessitates the development of clinical protocols to activate and expand to high numbers ex vivo to be able to infuse sufficient numbers of functional NK cells to the cancer patients. Initial NK cell-based clinical trials suggested that NK cell-infusion is safe and feasible with almost no NK cell-related toxicity, including graft-versus-host disease. Complete remission and increased disease-free survival is shown in a small number of patients with hematological malignances. Furthermore, successful adoptive NK cell-based therapies from haploidentical donors have been demonstrated. Disappointingly, only limited anti-tumor effects have been demonstrated following NK cell infusion in patients with solid tumors. While NK cells have great potential in targeting tumor cells, the efficiency of NK cell functions in the tumor microenvironment is yet unclear. The failure of immune surveillance may in part be due to sustained immunological pressure on tumor cells resulting in the development of tumor escape variants that are invisible to the immune system. Alternatively, this could be due to the complex network of immune-suppressive compartments in the tumor microenvironment, including myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Although the negative effect of the tumor microenvironment on NK cells can be transiently reverted by ex vivo expansion and long-term activation, the aforementioned NK cell/tumor microenvironment interactions upon reinfusion are not fully elucidated. Within this context, genetic modification of NK cells may provide new possibilities for developing effective cancer immunotherapies by improving NK cell responses and making them less susceptible to the tumor microenvironment. Within this review, we will discuss clinical trials using NK cells with a specific reflection on novel potential strategies, such as genetic modification of NK cells and complementary therapies aimed at improving the clinical outcome of NK cell-based immune therapies. PMID:26648934

Role of natural killer cells in lung cancer.

PubMed

Aktaş, Ozge Nur; Öztürk, Ayşe Bilge; Erman, Baran; Erus, Suat; Tanju, Serhan; Dilege, Şükrü

2018-06-01

One of the key immune cells involved in the pathogenesis of lung cancer is natural killer (NK) cells and these cells are novel targets for therapeutic applications in lung cancer. The purpose of this review is to summarize the current literature on lung cancer pathogenesis with a focus on the interaction between NK cells and smoking, how these factors are related to the pathogenesis of lung cancer and how NK cell-based immunotherapy effect lung cancer survival. The relevant literature from PubMed and Medline databases is reviewed in this article. The cytolytic potential of NK cells are reduced in lung cancer and increasing evidence suggests that improving NK cell functioning may induce tumor regression. Recent clinical trials on NK cell-based novel therapies such as cytokines including interleukin (IL)-15, IL-12 and IL-2, NK-92 cell lines and allogenic NK cell immunotherapy showed promising results with less adverse effects on the lung cancer survival. The NK cell targeting strategy has not yet been approved for lung cancer treatment. More clinical studies focusing on the role of NK cells in lung cancer pathogenesis are warranted to develop novel NK cell-based therapeutic approaches for the treatment of lung cancer.

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

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

Insufficient natural killer cell responses against retroviruses: how to improve NK cell killing of retrovirus-infected cells.

PubMed

Littwitz-Salomon, Elisabeth; Dittmer, Ulf; Sutter, Kathrin

2016-11-08

Natural killer (NK) cells belong to the innate immune system and protect against cancers and a variety of viruses including retroviruses by killing transformed or infected cells. They express activating and inhibitory receptors on their cell surface and often become activated after recognizing virus-infected cells. They have diverse antiviral effector functions like the release of cytotoxic granules, cytokine production and antibody dependent cellular cytotoxicity. The importance of NK cell activity in retroviral infections became evident due to the discovery of several viral strategies to escape recognition and elimination by NK cells. Mutational sequence polymorphisms as well as modulation of surface receptors and their ligands are mechanisms of the human immunodeficiency virus-1 to evade NK cell-mediated immune pressure. In Friend retrovirus infected mice the virus can manipulate molecular or cellular immune factors that in turn suppress the NK cell response. In this model NK cells lack cytokines for optimal activation and can be functionally suppressed by regulatory T cells. However, these inhibitory pathways can be overcome therapeutically to achieve full activation of NK cell responses and ultimately control dissemination of retroviral infection. One effective approach is to modulate the crosstalk between NK cells and dendritic cells, which produce NK cell-stimulating cytokines like type I interferons (IFN), IL-12, IL-15, and IL-18 upon retrovirus sensing or infection. Therapeutic administration of IFNα directly increases NK cell killing of retrovirus-infected cells. In addition, IL-2/anti-IL-2 complexes that direct IL-2 to NK cells have been shown to significantly improve control of retroviral infection by NK cells in vivo. In this review, we describe novel approaches to improve NK cell effector functions in retroviral infections. Immunotherapies that target NK cells of patients suffering from viral infections might be a promising treatment option for the future.

TAC1 Gene Products Regulate Pituitary Hormone Secretion and Gene Expression in Prepubertal Grass Carp Pituitary Cells.

PubMed

Hu, Guangfu; He, Mulan; Ko, Wendy K W; Wong, Anderson O L

2017-06-01

Tachykinin-1 (TAC1) is known to have diverse functions in mammals, but similar information is scarce in fish species. Using grass carp as a model, the pituitary actions, receptor specificity and postreceptor signaling of TAC1 gene products, namely substance P (SP) and neurokinin A (NKA), were examined. TAC1 encoding SP and NKA as well as tachykinin receptors NK1R and NK2R were cloned in the carp pituitary. The newly cloned receptors were shown to be functional with properties similar to mammalian counterparts. In carp pituitary cells, SP and NKA could trigger luteinizing hormone (LH), prolactin (PRL), and somatolactin α (SLα) secretion, with parallel rises in PRL and SLα transcripts. Short-term SP treatment (3 hours) induced LH release, whereas prolonged induction (24 hours) could attenuate LHβ messenger RNA (mRNA) expression. At pituitary cell level, LH, PRL, and SLα regulation by TAC1 gene products were mediated by NK1R, NK2R, and NK3R, respectively. Apparently, SP- and NKA-induced LH and SLα secretion and transcript expression were mediated by adenylyl cyclase/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), phospholiphase C (PLC)/inositol 1,4,5-triphosphate/protein kinase C (PKC), and Ca2+/calmodulin (CaM)/CaM-dependent protein kinase-II pathways. The signal transduction for PRL responses was similar, except for the absence of a PKC component. Regarding SP inhibition of LHβ mRNA expression, the cAMP/PKA- and PLC/PKC-dependent (but not Ca2+/CaM-dependent) cascades were involved. These results, as a whole, suggest that TAC1 gene products play a role in LH, PRL, and SLα regulation via overlapping postreceptor signaling coupled to different subtypes of tachykinin receptor expressed in the carp pituitary. Copyright © 2017 Endocrine Society.

Cytokines Induce Faster Membrane Diffusion of MHC Class I and the Ly49A Receptor in a Subpopulation of Natural Killer Cells

PubMed Central

Bagawath-Singh, Sunitha; Staaf, Elina; Stoppelenburg, Arie Jan; Spielmann, Thiemo; Kambayashi, Taku; Widengren, Jerker; Johansson, Sofia

2016-01-01

Cytokines have the potential to drastically augment immune cell activity. Apart from altering the expression of a multitude of proteins, cytokines also affect immune cell dynamics. However, how cytokines affect the molecular dynamics within the cell membrane of immune cells has not been addressed previously. Molecular movement is a vital component of all biological processes, and the rate of motion is, thus, an inherent determining factor for the pace of such processes. Natural killer (NK) cells are cytotoxic lymphocytes, which belong to the innate immune system. By fluorescence correlation spectroscopy, we investigated the influence of cytokine stimulation on the membrane density and molecular dynamics of the inhibitory receptor Ly49A and its ligand, the major histocompatibility complex class I allele H-2Dd, in freshly isolated murine NK cells. H-2Dd was densely expressed and diffused slowly in resting NK cells. Ly49A was expressed at a lower density and diffused faster. The diffusion rate in resting cells was not altered by disrupting the actin cytoskeleton. A short-term stimulation with interleukin-2 or interferon-α + β did not change the surface density of moving H-2Dd or Ly49A, despite a slight upregulation at the cellular level of H-2Dd by interferon-α + β, and of Ly49A by IL-2. However, the molecular diffusion rates of both H-2Dd and Ly49A increased significantly. A multivariate analysis revealed that the increased diffusion was especially marked in a subpopulation of NK cells, where the diffusion rate was increased around fourfold compared to resting NK cells. After IL-2 stimulation, this subpopulation of NK cells also displayed lower density of Ly49A and higher brightness per entity, indicating that Ly49A may homo-cluster to a larger extent in these cells. A faster diffusion of inhibitory receptors could enable a faster accumulation of these molecules at the immune synapse with a target cell, eventually leading to a more efficient NK cell response. It has previously been assumed that cytokines regulate immune cells primarily via alterations of protein expression levels or posttranslational modifications. These findings suggest that cytokines may also modulate immune cell efficiency by increasing the molecular dynamics early on in the response. PMID:26870035

Disordered expression of inhibitory receptors on the NK1-type natural killer (NK) leukaemic cells from patients with hypersensitivity to mosquito bites

PubMed Central

Seo, N; Tokura, Y; Ishihara, S; Takeoka, Y; Tagawa, S; Takigawa, M

2000-01-01

Recent studies have revealed the existence of a distinct type of NK cell leukaemia of the juvenile type, which presents with hypersensitivity to mosquito bites (HMB) as an essential clinical manifestation and is infected with clonal Epstein–Barr virus (EBV). This disorder is thus called HMB-EBV-NK disease and has been reported in Orientals, mostly from Japan. We investigated the profile of cytokine production and the expression of both types of NK inhibitory receptors, i.e. CD94 lectin-like dimers and killer-cell immunoglobulin-like receptors, in NK leukaemic cells from three patients with HMB-EBV-NK disease. It was found that freshly isolated NK leukaemic cells expressed mRNA for interferon-gamma (IFN-γ) and additionally produced IL-10 upon stimulation with IL-2, indicating that the NK cells were of NK1 type. More than 98% of NK cells from the patients bore CD94 at a higher level than did normal NK cells, whereas p70 or NKAT2, belonging to immunoglobulin-like receptor, was not expressed in those NK cells. Freshly isolated leukaemic NK cells transcribed mRNA for CD94-associated molecule NKG2C at an abnormally high level, and upon stimulation with IL-2 and/or IL-12 they expressed NKG2A as well. The disordered expression of these inhibitory receptors not only provides some insights into the pathogenesis of HMB-EBV-NK disease but also can be used as phenotypic markers for the diagnosis of this type of NK cell leukaemia. PMID:10844517

Biallelic mutations in IRF8 impair human NK cell maturation and function

PubMed Central

Mace, Emily M.; Gunesch, Justin T.; Chinn, Ivan K.; Angelo, Laura S.; Maisuria, Sheetal; Keller, Michael D.; Togi, Sumihito; Watkin, Levi B.; LaRosa, David F.; Jhangiani, Shalini N.; Muzny, Donna M.; Stray-Pedersen, Asbjørg; Coban Akdemir, Zeynep; Smith, Jansen B.; Hernández-Sanabria, Mayra; Le, Duy T.; Hogg, Graham D.; Cao, Tram N.; Freud, Aharon G.; Szymanski, Eva P.; Collin, Matthew; Cant, Andrew J.; Gibbs, Richard A.; Holland, Steven M.; Caligiuri, Michael A.; Ozato, Keiko; Paust, Silke; Doody, Gina M.; Lupski, James R.; Orange, Jordan S.

2016-01-01

Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8–/–, but not Irf8+/–, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense. PMID:27893462

Biallelic mutations in IRF8 impair human NK cell maturation and function.

PubMed

Mace, Emily M; Bigley, Venetia; Gunesch, Justin T; Chinn, Ivan K; Angelo, Laura S; Care, Matthew A; Maisuria, Sheetal; Keller, Michael D; Togi, Sumihito; Watkin, Levi B; LaRosa, David F; Jhangiani, Shalini N; Muzny, Donna M; Stray-Pedersen, Asbjørg; Coban Akdemir, Zeynep; Smith, Jansen B; Hernández-Sanabria, Mayra; Le, Duy T; Hogg, Graham D; Cao, Tram N; Freud, Aharon G; Szymanski, Eva P; Savic, Sinisa; Collin, Matthew; Cant, Andrew J; Gibbs, Richard A; Holland, Steven M; Caligiuri, Michael A; Ozato, Keiko; Paust, Silke; Doody, Gina M; Lupski, James R; Orange, Jordan S

2017-01-03

Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8-/-, but not Irf8+/-, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense.

The correlation between NK cell and liver function in patients with primary hepatocellular carcinoma.

PubMed

Sha, Wei Hong; Zeng, Xiao Hui; Min, Lu

2014-05-01

This study aimed to detect the expression of natural killer (NK) cell receptor natural killer group 2D (NKG2D) in the peripheral blood of patients with primary hepatocellular carcinoma and to discuss the correlation between NK cell cytotoxicity and liver function. The number of NK cells and the expression of NK cell receptor NKG2D in peripheral blood were determined by flow cytometry in patients with primary hepatocellular carcinoma, hepatitis B cirrhosis, chronic hepatitis B, and healthy controls. When compared with patients in the healthy and the chronic hepatitis B groups, the primary hepatocellular carcinoma group showed significant decreases in all parameters, including the cytotoxicity of NK cells on K562 cells, expression rate of NKG2D in NK cells, number of NKG2D(+) NK cells, expression level of NKG2D, and number of NK cells (p<0.05). The activity of NK cells showed a positive correlation, whereas the Child-Pugh scores in the primary hepatocellular carcinoma and the hepatitis B cirrhosis groups showed a negative correlation with all parameters detected above. The decrease of NK cell activity in patients with primary hepatocellular carcinoma is closely related to their lower expression of NKG2D. Liver function affects the expression of NKG2D and the activity of NK cells.

TLR8-driven IL-12-dependent reciprocal and synergistic activation of NK cells and monocytes by immunostimulatory RNA.

PubMed

Berger, Michael; Ablasser, Andrea; Kim, Sarah; Bekeredjian-Ding, Isabelle; Giese, Thomas; Endres, Stefan; Hornung, Veit; Hartmann, Gunther

2009-04-01

Immunostimulatory RNA (isRNA) depending on sequence and structure can function as a ligand for Toll-like receptor (TLR) 7 and TLR8. Here we show that isRNA induces high levels of bioactive interleukin-12 in purified human monocytes, whereas purified natural killer (NK) cells did not respond. However, in a coculture of monocytes and NK cells, isRNA dramatically increased NK cell function. Activation of monocytes and NK cells was bidirectional, as monocytes in the presence of NK cells produced higher levels of bioactive interleukin-12. As a result of the monocyte-NK cell interaction in peripheral blood mononuclear cells isRNA induced high levels of interferon (IFN)-gamma in NK cells and strong NK cell-mediated cytotoxic activity. Induction of simultaneous IFN-gamma production and lytic activity by isRNA in NK cells was higher as compared with other established nucleic acid or small molecule TLR ligands. Our studies demonstrate that monocytes play a pivotal role in the orchestration of a strong NK cell response. With early NK cell-dependent IFN-gamma production being critical for the development of antigen-specific cytotoxic T lymphocyte responses, newly developed isRNA-based TLR8 ligands join the list of promising oligonucleotides for immunotherapy of viral infection and cancer.

NK cell subsets in autoimmune diseases.

PubMed

Zhang, Cai; Tian, Zhigang

2017-09-01

Natural killer (NK) cells are lymphocytes of the innate immune system. They not only exert cell-mediated cytotoxicity against tumor cells or infected cells, but also play regulatory role through promoting or suppressing functions of other immune cells by secretion of cytokines and chemokines. However, overactivation or dysfunction of NK cells may be associated with pathogenesis of some diseases. NK cells are found to act as a two edged weapon and play opposite roles with both regulatory and inducer activity in autoimmune diseases. Though the precise mechanisms for the opposite effects of NK cells has not been fully elucidated, the importance of NK cells in autoimmune diseases might be associated with different NK cell subsets, different tissue microenvironment and different stages of corresponding diseases. The local tissue microenvironment, unique cellular interactions and different stages of corresponding diseases shape the properties and function of NK cells. In this review, we focus on recent research on the features and function of different NK cell subsets, particularly tissue-resident NK cells in different tissues, and their potential role in autoimmune diseases. Copyright © 2017. Published by Elsevier Ltd.

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells.

PubMed

Müller, Tina; Uherek, Christoph; Maki, Guitta; Chow, Kai Uwe; Schimpf, Annemarie; Klingemann, Hans-Georg; Tonn, Torsten; Wels, Winfried S

2008-03-01

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcgammaRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3zeta chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma.

PubMed

Sarkar, Saheli; Sabhachandani, Pooja; Ravi, Dashnamoorthy; Potdar, Sayalee; Purvey, Sneha; Beheshti, Afshin; Evens, Andrew M; Konry, Tania

2017-01-01

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

The anti-canine distemper virus activities of ex vivo-expanded canine natural killer cells.

PubMed

Park, Ji-Yun; Shin, Dong-Jun; Lee, Soo-Hyeon; Lee, Je-Jung; Suh, Guk-Hyun; Cho, Duck; Kim, Sang-Ki

2015-04-17

Natural killer (NK) cells play critical roles in induction of antiviral effects against various viruses of humans and animals. However, few data on NK cell activities during canine distemper virus (CDV) infections are available. Recently, we established a culture system allowing activation and expansion of canine non-B, non-T, large granular NK lymphocytes from PBMCs of normal dogs. In the present study, we explored the ability of such expanded NK cells to inhibit CDV infection in vitro. Cultured CD3-CD5-CD21- NK cells produced large amounts of IFN-γ, exhibited highly upregulated expression of mRNAs encoding NK-cell-associated receptors, and demonstrated strong natural killing activity against canine tumor cells. Although the expanded NK cells were dose-dependently cytotoxic to both normal and CDV-infected Vero cells, CDV infection rendered Vero cells more susceptible to NK cells. Pretreatment with anti-CDV serum from hyperimmunized dogs enhanced the antibody-dependent cellular cytotoxicity (ADCC) of NK cells against CDV-infected Vero cells. The culture supernatants of NK cells, added before or after infection, dose-dependently inhibited both CDV replication and development of CDV-induced cytopathic effects (CPEs) in Vero cells. Anti-IFN-γ antibody neutralized the inhibitory effects of NK cell culture supernatants on CDV replication and CPE induction in Vero cells. Such results emphasize the potential significance of NK cells in controlling CDV infection, and indicate that NK cells may play roles both during CDV infection and in combating such infections, under certain conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Baculovirus directly activates murine NK cells via TLR9.

PubMed

Moriyama, T; Suzuki, T; Chang, M O; Kitajima, M; Takaku, H

2017-04-01

The importance of natural killer (NK) cells in innate immune responses against tumors or viral infections enhances the appeal of NK cell-based immunotherapeutic approaches. We have recently reported that baculovirus (BV)-infected dendritic cells (DCs; BV-DCs) induce antitumor immunity against established tumors in mice. These antitumor effects were CD8 + T-cell and NK cell dependent; however, they were found to be CD4 + T-cell independent. In this study, we investigated the involvement of Toll-like receptor 9 (TLR9) in the process of BV recognition by NK cells. We found that BV directly stimulated NK cells, induced the expression of the activation marker CD69 and promoted interferon-gamma (IFN-γ) production and cytotoxicity. Moreover, TLR9 knockout in mice (tlr9-/- NK cells) inhibited NK cell responses to BV, indicating that TLR9 may have a relevant role in the BV-induced upregulation of NK cell functions. Our data demonstrated for the first time that NK cells directly recognize BV via TLR9, which provides opportunities for the use of this technique as an effective tool for BV-based immunotherapies against malignancies.

Natural killer cell biology illuminated by primary immunodeficiency syndromes in humans.

PubMed

Voss, Matthias; Bryceson, Yenan T

2017-04-01

Natural killer (NK) cells are innate immune cytotoxic effector cells well known for their role in antiviral immunity and tumor immunosurveillance. In parts, this knowledge stems from rare inherited immunodeficiency disorders in humans that abrogate NK cell function leading to immune impairments, most notably associated with a high susceptibility to viral infections. Phenotypically, these disorders range from deficiencies selectively affecting NK cells to complex general immune defects that affect NK cells but also other immune cell subsets. Moreover, deficiencies may be associated with reduced NK cell numbers or rather impair specific NK cell effector functions. In recent years, genetic defects underlying the various NK cell deficiencies have been uncovered and have triggered investigative efforts to decipher the molecular mechanisms underlying these disorders. Here we review the associations between inherited human diseases and NK cell development as well as function, with a particular focus on defects in NK cell exocytosis and cytotoxicity. Furthermore we outline how reports of diverse genetic defects have shaped our understanding of NK cell biology. Copyright © 2015. Published by Elsevier Inc.

Increased IFN-gamma production by NK and CD3+/CD56+ cells in sexually HIV-1-exposed but uninfected individuals.

PubMed

Montoya, Carlos Julio; Velilla, Paula Andrea; Chougnet, Claire; Landay, Alan L; Rugeles, Maria Teresa

2006-08-01

The mechanisms involved in controlling the establishment of HIV-1 infection are not fully understood. In particular, the role of innate immunity in natural resistance exhibited by individuals who are continuously exposed to HIV-1 but remain seronegative (ESN) has not been thoroughly evaluated. We determined the frequency and function of peripheral blood innate immune cells (plasmacytoid and myeloid dendritic cells, monocytes, NK cells, CD3+/CD56+ cells and invariant NKT cells) in ESN, chronically HIV-1-infected and low-risk HIV-1 seronegative individuals. ESN demonstrated a similar frequency of innate immune cells in comparison to controls and a higher frequency of dendritic cells, NK and invariant NKT cells compared to HIV-1-infected subjects. Incubation of mononuclear cells with stimulatory CpG ODN induced CD86 and CD69 up-regulation to a similar degree on innate cells from the three study groups. CpG ODN-stimulated secretion of cytokines was also similar between ESN and controls, while secretion of IFN-alpha was significantly decreased in HIV-1+ individuals. Importantly, expression of IFN-gamma by PMA/Ionomycin-activated CD56(bright) NK cells and CD3+/CD56+ cells was significantly higher in ESN when compared with controls. The anti-viral effects of IFN-gamma are well established, and so our results suggest that IFN-gamma production by innate immune cells might be one of the multiple factors involved in controlling the establishment of sexually transmitted HIV-1 infection.

Ex vivo expanded natural killer cells from breast cancer patients and healthy donors are highly cytotoxic against breast cancer cell lines and patient-derived tumours.

PubMed

Shenouda, Mira M; Gillgrass, Amy; Nham, Tina; Hogg, Richard; Lee, Amanda J; Chew, Marianne V; Shafaei, Mahsa; Aarts, Craig; Lee, Dean A; Hassell, John; Bane, Anita; Dhesy-Thind, Sukhbinder; Ashkar, Ali A

2017-07-01

Natural killer (NK) cells play a critical role in cancer immunosurveillance. Recent developments in NK cell ex-vivo expansion makes it possible to generate millions of activated NK cells from a small volume of peripheral blood. We tested the functionality of ex vivo expanded NK cells in vitro against breast cancer cell lines and in vivo using a xenograft mouse model. The study aim was to assess functionality and phenotype of expanded NK cells from breast cancer patients against breast cancer cell lines and autologous primary tumours. We used a well-established NK cell co-culture system to expand NK cells ex vivo from healthy donors and breast cancer patients and examined their surface marker expression. Moreover, we tested the ability of expanded NK cells to lyse the triple negative breast cancer and HER2-positive breast cancer cell lines MDA-MB-231 and MDA-MB-453, respectively. We also tested their ability to prevent tumour growth in vivo using a xenograft mouse model. Finally, we tested the cytotoxicity of expanded NK cells against autologous and allogeneic primary breast cancer tumours in vitro. After 3 weeks of culture we observed over 1000-fold expansion of NK cells isolated from either breast cancer patients or healthy donors. We also showed that the phenotype of expanded NK cells is comparable between those from healthy donors and cancer patients. Moreover, our results confirm the ability of ex vivo expanded NK cells to lyse tumour cell lines in vitro. While the cell lines examined had differential sensitivity to NK cell killing we found this was correlated with level of major histocompatibility complex (MHC) class I expression. In our in vivo model, NK cells prevented tumour establishment and growth in immunocompromised mice. Finally, we showed that NK cells expanded from the peripheral blood of breast cancer patients show high cytotoxicity against allogeneic and autologous patient-derived tumour cells in vitro. NK cells from breast cancer patients can be expanded similarly to those from healthy donors, have a high cytotoxic ability against breast cancer cell lines and patient-derived tumour cells, and can be compatible with current cancer treatments to restore NK cell function in cancer patients.

Generation of natural killer cells from hematopoietic stem cells in vitro for immunotherapy

PubMed Central

Luevano, Martha; Madrigal, Alejandro; Saudemont, Aurore

2012-01-01

Natural killer (NK) cells are part of the innate immune system and are an alluring option for immunotherapy due to their ability to kill infected cells or cancer cells without prior sensitization. Throughout the past 20 years, different groups have been able to reproduce NK cell development in vitro, and NK cell ontogeny studies have provided the basis for the establishment of protocols to produce NK cells in vitro for immunotherapy. Here, we briefly discuss NK cell development and NK cell immunotherapy approaches. We review the factors needed for NK cell differentiation in vitro, which stem cell sources have been used, published protocols, challenges and future directions for Good Manufacturing Practice protocols. PMID:22705914

NK Cells, Tumor Cell Transition, and Tumor Progression in Solid Malignancies: New Hints for NK-Based Immunotherapy?

PubMed Central

Huergo-Zapico, Leticia; Parodi, Monica; Pedrazzi, Marco; Mingari, Maria Cristina; Sparatore, Bianca; Gonzalez, Segundo; Olive, Daniel; Bottino, Cristina

2016-01-01

Several evidences suggest that NK cells can patrol the body and eliminate tumors in their initial phases but may hardly control established solid tumors. Multiple factors, including the transition of tumor cells towards a proinvasive/prometastatic phenotype, the immunosuppressive effect of the tumor microenvironment, and the tumor structure complexity, may account for limited NK cell efficacy. Several putative mechanisms of NK cell suppression have been defined in these last years; conversely, the cross talk between NK cells and tumor cells undergoing different transitional phases remains poorly explored. Nevertheless, recent in vitro studies and immunohistochemical analyses on tumor biopsies suggest that NK cells could not only kill tumor cells but also influence their evolution. Indeed, NK cells may induce tumor cells to change the expression of HLA-I, PD-L1, or NKG2D-L and modulate their susceptibility to the immune response. Moreover, NK cells may be preferentially located in the borders of tumor masses, where, indeed, tumor cells can undergo Epithelial-to-Mesenchymal Transition (EMT) acquiring prometastatic phenotype. Finally, the recently highlighted role of HMGB1 both in EMT and in amplifying the recruitment of NK cells provides further hints on a possible effect of NK cells on tumor progression and fosters new studies on this issue. PMID:27294158

Revving up Natural Killer Cells and Cytokine-Induced Killer Cells Against Hematological Malignancies.

PubMed

Pittari, Gianfranco; Filippini, Perla; Gentilcore, Giusy; Grivel, Jean-Charles; Rutella, Sergio

2015-01-01

Natural killer (NK) cells belong to innate immunity and exhibit cytolytic activity against infectious pathogens and tumor cells. NK-cell function is finely tuned by receptors that transduce inhibitory or activating signals, such as killer immunoglobulin-like receptors, NK Group 2 member D (NKG2D), NKG2A/CD94, NKp46, and others, and recognize both foreign and self-antigens expressed by NK-susceptible targets. Recent insights into NK-cell developmental intermediates have translated into a more accurate definition of culture conditions for the in vitro generation and propagation of human NK cells. In this respect, interleukin (IL)-15 and IL-21 are instrumental in driving NK-cell differentiation and maturation, and hold great promise for the design of optimal NK-cell culture protocols. Cytokine-induced killer (CIK) cells possess phenotypic and functional hallmarks of both T cells and NK cells. Similar to T cells, they express CD3 and are expandable in culture, while not requiring functional priming for in vivo activity, like NK cells. CIK cells may offer some advantages over other cell therapy products, including ease of in vitro propagation and no need for exogenous administration of IL-2 for in vivo priming. NK cells and CIK cells can be expanded using a variety of clinical-grade approaches, before their infusion into patients with cancer. Herein, we discuss GMP-compliant strategies to isolate and expand human NK and CIK cells for immunotherapy purposes, focusing on clinical trials of adoptive transfer to patients with hematological malignancies.

Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunologic synapses

NASA Astrophysics Data System (ADS)

Orange, Jordan S.; Ramesh, Narayanaswamy; Remold-O'Donnell, Eileen; Sasahara, Yoji; Koopman, Louise; Byrne, Michael; Bonilla, Francisco A.; Rosen, Fred S.; Geha, Raif S.; Strominger, Jack L.

2002-08-01

The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by a mutation in WAS protein (WASp) that results in defective actin polymerization. Although the function of many hematopoietic cells requires WASp, the specific expression and function of this molecule in natural killer (NK) cells is unknown. Here, we report that WAS patients have increased percentages of peripheral blood NK cells and that fresh enriched NK cells from two patients with a WASp mutation have defective cytolytic function. In normal NK cells, WASp was expressed and localized to the activating immunologic synapse (IS) with filamentous actin (F-actin). Perforin also localized to the NK cell-activating IS but at a lesser frequency than F-actin and WASp. The accumulation of F-actin and WASp at the activating IS was decreased significantly in NK cells that had been treated with the inhibitor of actin polymerization, cytochalasin D. NK cells from WAS patients lacked expression of WASp and accumulated F-actin at the activating IS infrequently. Thus, WASp has an important function in NK cells. In patients with WASp mutations, the resulting NK cell defects are likely to contribute to their disease.

KHYG-1 and NK-92 represent different subtypes of LFA-1-mediated NK cell adhesiveness.

PubMed

Suck, Garnet; Tan, Suet-Mien; Chu, Sixian; Niam, Madelaine; Vararattanavech, Ardcharaporn; Lim, Tsyr Jong; Koh, Mickey B C

2011-01-01

Novel cancer cellular therapy approaches involving long-term ex vivo IL-2 stimulated highly cytotoxic natural killer (NK) cells are emerging. However, adhesion properties of such NK cells are not very well understood. Herein, we describe the novel observation of permanently activated alphaLbeta2 integrin leukocyte function-associated antigen (LFA)-1 adhesion receptor in long-term IL-2 activated NK cells and the permanent NK cell lines KHYG-1 and NK-92. We show that such cytokine activated NK effectors constitutively adhered to the LFA-1-ligand ICAM-1, whereas binding to the lower affinity ligand ICAM-3 required additional exogenous activating conditions. The results demonstrate an extended conformation and an intermediate affinity state for the LFA-1 population expressed by the NK cells. Interestingly, adhesion to ICAM-1 or K562 induced pronounced cell spreading in KHYG-1, but not in NK-92, and partially in long-term IL-2 stimulated primary NK cells. It is conceivable that such differential adhesion characteristics may impact motility potential of such NK effectors with relevance to clinical tumor targeting. KHYG-1 could be a useful model in planning future targeted therapeutic approaches involving NK effectors with augmented functions.

Cell killing mode of liblomycin (NK313), a novel dose-survival relationship different from bleomycins.

PubMed

Kuramochi-Motegi, A; Kuramochi, H; Takahashi, K; Takeuchi, T

1991-04-01

Liblomycin (NK313) is a novel derivative of bleomycin (BLM) and peplomycin (PEP). The cell kill kinetics of NK313 on rat ascites hepatoma AH66 were compared with those of PEP. NK313 induced intracellular DNA cleavage and arrested cell cycle progression at the G2 phase similarly to PEP. The cytocidal effect of NK313, however, was found to be different from that of PEP as described below: 1) The dose-survival curve for cells exposed to PEP for 1 hour was upward concave, whereas in case of NK313, the survival curve was linear. PEP was more effective to AH66 than NK313 at lower concentration, but at higher concentration, NK313 was much more effective. 2) The time-survival curve for cells treated with either NK313 or PEP was biphasic. NK313, however, did not induce temporary resistance of AH66 cells to NK313, while PEP induced resistance to PEP. 3) NK313 was effective against the cells which became temporarily resistant to PEP by the treatment of PEP. These differences suggest that NK313 might be of value to treat PEP-insensitive tumor cells.

Effect of CMV and Aging on the Differential Expression of CD300a, CD161, T-bet, and Eomes on NK Cell Subsets.

PubMed

Lopez-Sejas, Nelson; Campos, Carmen; Hassouneh, Fakhri; Sanchez-Correa, Beatriz; Tarazona, Raquel; Pera, Alejandra; Solana, Rafael

2016-01-01

Natural killer (NK) cells are innate lymphoid cells involved in the defense against virus-infected cells and tumor cells. NK cell phenotype and function is affected with age and cytomegalovirus (CMV) latent infection. Aging affects the frequency and phenotype of NK cells, and CMV infection also contributes to these alterations. Thus, a reduction of CD56 bright NK cell subpopulation associated with age and an expansion of memory-like NK cells CD56 dim CD57 + NKG2C + probably related to CMV seropositivity have been described. NK cells express T-bet and Eomes transcription factors that are necessary for the development of NK cells. Here, we analyze the effect of age and CMV seropositivity on the expression of CD300a and CD161 inhibitory receptors, and T-bet and Eomes transcription factors in NK cell subsets defined by the expression of CD56 and CD57. CD300a is expressed by the majority of NK cells. CD56 bright NK cells express higher levels of CD300a than CD56 dim NK cells. An increase in the expression of CD300a was associated with age, whereas a decreased expression of CD161 in CD56 dim NK cells was associated with CMV seropositivity. In CD56 dim NK cells, an increased percentage of CD57 + CD300a + and a reduction in the percentage of CD161 + CD300a + cells were found to be associated with CMV seropositivity. Regarding T-bet and Eomes transcription factors, CMV seropositivity was associated with a decrease of T-bet hi in CD56 dim CD57 + NK cells from young individuals, whereas Eomes expression was increased with CMV seropositivity in both CD56 bright and CD56 dim CD57 +/- (from middle age and young individuals, respectively) and was decreased with aging in all NK subsets from the three group of age. In conclusion, CMV infection and age induce significant changes in the expression of CD300a and CD161 in NK cell subsets defined by the expression of CD56 and CD57. T-bet and Eomes are differentially expressed on NK cell subsets, and their expression is affected by CMV latent infection and aging.

Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity.

PubMed

Zhang, Congcong; Oberoi, Pranav; Oelsner, Sarah; Waldmann, Anja; Lindner, Aline; Tonn, Torsten; Wels, Winfried S

2017-01-01

Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR-engineered NK-92 cells as off-the-shelf cellular therapeutics, with special emphasis on ErbB2 (HER2)-specific NK-92 cells that are approaching clinical application.

Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity

PubMed Central

Zhang, Congcong; Oberoi, Pranav; Oelsner, Sarah; Waldmann, Anja; Lindner, Aline; Tonn, Torsten; Wels, Winfried S.

2017-01-01

Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR-engineered NK-92 cells as off-the-shelf cellular therapeutics, with special emphasis on ErbB2 (HER2)-specific NK-92 cells that are approaching clinical application. PMID:28572802

Development of IL-22–producing NK lineage cells from umbilical cord blood hematopoietic stem cells in the absence of secondary lymphoid tissue

PubMed Central

Tang, Qin; Ahn, Yong-Oon; Southern, Peter; Blazar, Bruce R.; Miller, Jeffery S.

2011-01-01

Human secondary lymphoid tissues (SLTs) contain interleukin-22 (IL-22)–producing cells with an immature NK phenotype. Given their location, these cells are difficult to study. We have generated large numbers of NK22 cells from hematopoietic stem cells. HSC-derived NK22 cells show a CD56+CD117highCD94− phenotype, consistent with stage III NK progenitors. Like freshly isolated SLT stage III cells, HSC-derived NK22 cells express NKp44, CD161, CCR6, IL1 receptor, AHR, and ROR-γτ. IL-1β and IL-23 stimulation results in significant IL-22 but not interferon-γ production. Supernatant from these cells increases CD54 expression on mesenchymal stem cells. Thus, IL-22–producing NK cells can be generated in the absence of SLT. HSC-derived NK22 cells will be valuable in understanding this rare NK subset and create the opportunity for human translational clinical trials. PMID:21310921

Development of IL-22-producing NK lineage cells from umbilical cord blood hematopoietic stem cells in the absence of secondary lymphoid tissue.

PubMed

Tang, Qin; Ahn, Yong-Oon; Southern, Peter; Blazar, Bruce R; Miller, Jeffery S; Verneris, Michael R

2011-04-14

Human secondary lymphoid tissues (SLTs) contain interleukin-22 (IL-22)-producing cells with an immature NK phenotype. Given their location, these cells are difficult to study. We have generated large numbers of NK22 cells from hematopoietic stem cells. HSC-derived NK22 cells show a CD56(+)CD117(high)CD94(-) phenotype, consistent with stage III NK progenitors. Like freshly isolated SLT stage III cells, HSC-derived NK22 cells express NKp44, CD161, CCR6, IL1 receptor, AHR, and ROR-γτ. IL-1β and IL-23 stimulation results in significant IL-22 but not interferon-γ production. Supernatant from these cells increases CD54 expression on mesenchymal stem cells. Thus, IL-22-producing NK cells can be generated in the absence of SLT. HSC-derived NK22 cells will be valuable in understanding this rare NK subset and create the opportunity for human translational clinical trials.

Umbilical cord blood-derived natural killer cells combined with Bevacizumab for colorectal cancer treatment.

PubMed

Xu, Chen; Liu, Dongning; Chen, Zhixin; Zhuo, Fan; Sun, Huankui; Hu, Jiaping; Li, Taiyuan

2018-06-19

Colorectal cancer (CRC) is among cancers with highest incidence globally and currently ranks fourth as the leading cause of cancer-related deaths worldwide. It remains an urgent need for novel strategies in the management of patients with advanced CRC. Adoptive transfer of allogeneic natural killer (NK) cells represent an attractive option in the treatment of patients with CRC. In this study, we successfully expanded NK cells from umbilical cord blood (UCB) with membrane-bound IL-21, termed eUCB-NK cells. eUCB-NK cells efficiently lysed CRC cell lines in vitro and secreted significantly higher levels of IFN-γ, TNF-α, GM-CSF and CCL3 compared with IL-2 stimulated NK cells. Adoptive transfer of these NK cells significantly inhibited the growth of HT29 xenografts, whereas LoVo tumors were not effectively controlled with eUCB-NK cells. More NK cells inside HT29 tumors, not seen in LoVo tumors, might contribute to the differences in response to eUCB-NK cells. Combination of bevacizumab can increase extravasation of adoptively transferred NK cells into the LoVo tumors and improve the therapeutic activity of eUCB-NK cells. These results justified clinical translation of this UCB-derived NK cell-based therapeutics, either used alone or combined with bevacizumab, as a novel treatment option for patients with CRC.

Gene delivery of TIPE2 inhibits breast cancer development and metastasis via CD8+ T and NK cell-mediated antitumor responses.

PubMed

Zhang, Zhenhua; Liu, Li; Cao, Shousong; Zhu, Yizhun; Mei, Qibing

2017-05-01

Breast cancer is the second leading cause of cancer-related deaths in the female patients which was mainly caused by metastasis. Development of target gene therapy for breast cancer to suppress tumor progress and metastasis will improve the therapeutic options and be of great benefit to the patients. Tumor necrosis factor-alpha-induced protein 8-like 2 is a novel molecule for maintaining immune homeostasis and is involved in cancer development. In the present study, we overexpressed TIPE2 in breast cancer cells to investigate the role of TIPE2 in the development of breast cancer. Our results showed that overexpression of TIPE2 significantly inhibited the proliferation of 4T1 cells in vitro and in vivo. We constructed a non-viral targeted gene therapeutic system by using the minicircle plasmids expressing TIPE2. We found that the growth and metastasis of breast cancer was significantly inhibited by hydrodynamic gene delivery of TIPE2 plasmids in vivo. Mechanistically, TIPE2 increased T and NK cells, and decreased MDSCs. Gene delivery of TIPE2 up-regulated the production of IFN-γ and TNF-α by CD8 + T and NK cells in spleens and tumor microenvironment, and enhanced the cytotoxic activity of CD8 + T and NK cells. Taken together, TIPE2 inhibited breast cancer development and metastasis possibly via promoting CD8 + T and NK cell-mediated antitumor immune responses. Thus, the results indicate that TIPE2 may be a potential therapeutic target for breast cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Formaldehyde exposure impairs the function and differentiation of NK cells.

PubMed

Kim, Eun-Mi; Lee, Hwa-Youn; Lee, Eun-Hee; Lee, Ki-Mo; Park, Min; Ji, Kon-Young; Jang, Ji-Hun; Jeong, Yun-Hwa; Lee, Kwang-Ho; Yoon, Il-Joo; Kim, Su-Man; Jeong, Moon-Jin; Kim, Kwang Dong; Kang, Hyung-Sik

2013-11-25

We investigated the cytotoxic effects of formaldehyde (FA) on lymphocytes. FA-exposed mice showed a profound reduction not only in the number of natural killer (NK) cells but also in the expression of NK cell-specific receptors, but these mice did not exhibit decreases in the numbers of T or B lymphocytes. FA exposure also induced decreases in NK cytolytic activity and in the expression of NK cell-associated genes, such as IFN-γ, perforin and CD122. To determine the effect of FA on tumorigenicity, C57BL/6 mice were subcutaneously injected with B16F10 melanoma cells after FA exposure. The mass of the B16F10 tumor and the concentration of extravascular polymorphonuclear leukocytes were greater than those in unexposed tumor-bearing control mice. The number and cytolytic activity of NK cells were also reduced in B16F10 tumor-bearing mice exposed to FA. To determine how FA reduces the NK cell number, NK precursor (pNK) cells were treated with FA, and the differentiation status of the NK cells was analyzed. NK cell differentiation was impaired by FA treatment in a concentration-dependent manner. These findings indicate that FA exposure may promote tumor progression by impairing NK cell function and differentiation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

Miranda, Dante; Jara, Claudia; Mejias, Sophia; Ahumada, Viviana; Cortez-San Martin, Marcelo; Ibañez, Jorge; Hirsch, Sandra; Montoya, Margarita

2018-05-18

Immunosenescence has been described as age-associated changes in the immune function which are thought to be responsible for the increased morbidity with age. Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in immune defense against tumor and microbial diseases. Interestingly, aging-related NK cell dysfunction is associated with features of aging such as tumor incidence, reduced vaccination efficacy, and short survival due to infection. It is known that NK cell effector functions are critically dependent on cytokines and metabolic activity. Our aim was to determine whether there is a difference in purified human NK cell function in response to high concentration of IL-2 between young and elder donors. Here, we report that the stimulation of human NK cells with IL-2 (2000 U/mL) enhance NK cell cytotoxic activity from both young and elderly donors. However, while NK cells from young people responded to IL-2 signaling by increasing mitochondrial mass and mitochondrial membrane potential, no increase in these mitochondrial functional parameters was seen in purified NK cells from elderly subjects. Moreover, as purified NK cells from the young exhibited an almost three-fold increase in PGC-1α expression after IL-2 (2000 U/mL) stimulation, PGC-1α expression was inhibited in purified NK cells from elders. Furthermore, this response upon PGC-1α expression after IL-2 stimulation promoted an increase in ROS production in NK cells from elderly humans, while no increase in ROS production was observed in NK cells of young donors. Our data show that IL-2 stimulates NK cell effector function through a signaling pathway which involves a PGC-1α-dependent mitochondrial function in young NK cells, however it seems that NK cells from older donors exhibit an altered IL-2 signaling which affects mitochondrial function associated with an increased production of ROS which could represent a feature of NK cell senescence. Copyright © 2018 Elsevier Inc. All rights reserved.

IL‐12 and IL‐15 induce the expression of CXCR6 and CD49a on peripheral natural killer cells

PubMed Central

Hydes, Theresa; Noll, Angela; Salinas‐Riester, Gabriela; Abuhilal, Mohammed; Armstrong, Thomas; Hamady, Zaed; Primrose, John; Takhar, Arjun; Walter, Lutz

2017-01-01

Abstract Introduction Murine hepatic NK cells exhibit adaptive features, with liver‐specific adhesion molecules CXCR6 and CD49a acting as surface markers. Methods We investigated human liver‐resident CXCR6+ and CD49a+ NK cells using RNA sequencing, flow cytometry, and functional analysis. We further assessed the role of cytokines in generating NK cells with these phenotypes from the peripheral blood. Results Hepatic CD49a+ NK cells could be induced using cytokines and produce high quantities of IFNγ and TNFα, in contrast to hepatic CXCR6+ NK cells. RNA sequencing of liver‐resident CXCR6+ NK cells confirmed a tolerant immature phenotype with reduced expression of markers associated with maturity and cytotoxicity. Liver‐resident double‐positive CXCR6 + CD49a+ hepatic NK cells are immature but maintain high expression of Th1 cytokines as observed for single‐positive CD49a+ NK cells. We show that stimulation with activating cytokines can readily induce upregulation of both CD49a and CXCR6 on NK cells in the peripheral blood. In particular, IL‐12 and IL‐15 can generate CXCR6 + CD49a+ NK cells in vitro from NK cells isolated from the peripheral blood, with comparable phenotypic and functional features to liver‐resident CD49a+ NK cells, including enhanced IFNγ and NKG2C expression. Conclusion IL‐12 and IL‐15 may be key for generating NK cells with a tissue‐homing phenotype and strong Th1 cytokine profile in the blood, and links peripheral activation of NK cells with tissue‐homing. These findings may have important therapeutic implications for immunotherapy of chronic liver disease. PMID:28952190

IL-12 and IL-15 induce the expression of CXCR6 and CD49a on peripheral natural killer cells.

PubMed

Hydes, Theresa; Noll, Angela; Salinas-Riester, Gabriela; Abuhilal, Mohammed; Armstrong, Thomas; Hamady, Zaed; Primrose, John; Takhar, Arjun; Walter, Lutz; Khakoo, Salim I

2018-03-01

Murine hepatic NK cells exhibit adaptive features, with liver-specific adhesion molecules CXCR6 and CD49a acting as surface markers. We investigated human liver-resident CXCR6+ and CD49a+ NK cells using RNA sequencing, flow cytometry, and functional analysis. We further assessed the role of cytokines in generating NK cells with these phenotypes from the peripheral blood. Hepatic CD49a+ NK cells could be induced using cytokines and produce high quantities of IFNγ and TNFα, in contrast to hepatic CXCR6+ NK cells. RNA sequencing of liver-resident CXCR6+ NK cells confirmed a tolerant immature phenotype with reduced expression of markers associated with maturity and cytotoxicity. Liver-resident double-positive CXCR6 + CD49a+ hepatic NK cells are immature but maintain high expression of Th1 cytokines as observed for single-positive CD49a+ NK cells. We show that stimulation with activating cytokines can readily induce upregulation of both CD49a and CXCR6 on NK cells in the peripheral blood. In particular, IL-12 and IL-15 can generate CXCR6 + CD49a+ NK cells in vitro from NK cells isolated from the peripheral blood, with comparable phenotypic and functional features to liver-resident CD49a+ NK cells, including enhanced IFNγ and NKG2C expression. IL-12 and IL-15 may be key for generating NK cells with a tissue-homing phenotype and strong Th1 cytokine profile in the blood, and links peripheral activation of NK cells with tissue-homing. These findings may have important therapeutic implications for immunotherapy of chronic liver disease. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients.

PubMed

Siegler, Uwe; Meyer-Monard, Sandrine; Jörger, Simon; Stern, Martin; Tichelli, André; Gratwohl, Alois; Wodnar-Filipowicz, Aleksandra; Kalberer, Christian P

2010-10-01

Alloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use. We describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells. NK cells (6.0 ± 1.2 x 10(8)) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR+ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML. The approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.

Involvement of substance P and the NK-1 receptor in human pathology.

PubMed

Muñoz, Miguel; Coveñas, Rafael

2014-07-01

The peptide substance P (SP) shows a widespread distribution in both the central and peripheral nervous systems, but it is also present in cells not belonging to the nervous system (immune cells, liver, lung, placenta, etc.). SP is located in all body fluids, such as blood, cerebrospinal fluid, breast milk, etc. i.e. it is ubiquitous in human body. After binding to the neurokinin-1 (NK-1) receptor, SP regulates many pathophysiological functions in the central nervous system, such as emotional behavior, stress, depression, anxiety, emesis, vomiting, migraine, alcohol addiction, seizures and neurodegeneration. SP has been also implicated in pain, inflammation, hepatitis, hepatotoxicity, cholestasis, pruritus, myocarditis, bronchiolitis, abortus, bacteria and viral infection (e.g., HIV infection) and it plays an important role in cancer (e.g., tumor cell proliferation, antiapoptotic effects in tumor cells, angiogenesis, migration of tumor cells for invasion, infiltration and metastasis). This means that the SP/NK-1 receptor system is involved in the molecular bases of many human pathologies. Thus, knowledge of this system is the key for a better understanding and hence a better management of many human diseases. In this review, we update the involvement of the SP/NK-1 receptor system in the physiopathology of the above-mentioned pathologies and we suggest valuable future therapeutic interventions involving the use of NK-1 receptor antagonists, particularly in the treatment of emesis, depression, cancer, neural degeneration, inflammatory bowel disease, viral infection and pruritus, in which that system is upregulated.

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

2016-04-04

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, radiolabeled NK cells were retained in the lung tumor lesions up to 72 h p.i. without tumor regression. In tumor-bearing mice that were only irradiated but did not receive radiolabeled murine NK cells, a high tumor burden was observed at 72 h p.i., which indicates that irradiation in combination with murine NK cell allocation, but not irradiation alone, induced a remarkable antitumor effect in the orthotopic A549 lung tumor bearing mouse model. In conclusion, we describe a method to evaluate murine NK cell trafficking and biodistribution, which can be used to determine potential effects of immune-mediated therapeutic agents on NK cell biodistribution.

Overexpression of membrane metalloendopeptidase inhibits substance P stimulation of cholangiocarcinoma growth.

PubMed

Meng, Fanyin; DeMorrow, Sharon; Venter, Julie; Frampton, Gabriel; Han, Yuyan; Francis, Heather; Standeford, Holly; Avila, Shanika; McDaniel, Kelly; McMillin, Matthew; Afroze, Syeda; Guerrier, Micheleine; Quezada, Morgan; Ray, Debolina; Kennedy, Lindsey; Hargrove, Laura; Glaser, Shannon; Alpini, Gianfranco

2014-05-01

Substance P (SP) promotes cholangiocyte growth during cholestasis by activating its receptor, NK1R. SP is a proteolytic product of tachykinin (Tac1) and is deactivated by membrane metalloendopeptidase (MME). This study aimed to evaluate the functional role of SP in the regulation of cholangiocarcinoma (CCA) growth. NK1R, Tac1, and MME expression and SP secretion were assessed in human CCA cells and nonmalignant cholangiocytes. The proliferative effects of SP (in the absence/presence of the NK1R inhibitor, L-733,060) and of L-733,060 were evaluated. In vivo, the effect of L-733,060 treatment or MME overexpression on tumor growth was evaluated by using a xenograft model of CCA in nu/nu nude mice. The expression of Tac1, MME, NK1R, PCNA, CK-19, and VEGF-A was analyzed in the resulting tumors. Human CCA cell lines had increased expression of Tac1 and NK1R, along with reduced levels of MME compared with nonmalignant cholangiocytes, resulting in a subsequent increase in SP secretion. SP treatment increased CCA cell proliferation in vitro, which was blocked by L-733,060. Treatment with L-733,060 alone inhibited CCA proliferation in vitro and in vivo. Xenograft tumors derived from MME-overexpressed human Mz-ChA-1 CCA cells had a slower growth rate than those derived from control cells. Expression of PCNA, CK-19, and VEGF-A decreased, whereas MME expression increased in the xenograft tumors treated with L-733,060 or MME-overexpressed xenograft tumors compared with controls. The study suggests that SP secreted by CCA promotes CCA growth via autocrine pathway. Blockade of SP secretion and NK1R signaling may be important for the management of CCA.

Eomeshi NK Cells in Human Liver Are Long-Lived and Do Not Recirculate but Can Be Replenished from the Circulation

PubMed Central

Cuff, Antonia O.; Robertson, Francis P.; Stegmann, Kerstin A.; Pallett, Laura J.; Maini, Mala K.; Davidson, Brian R.

2016-01-01

Human liver contains an Eomeshi population of NK cells that is not present in the blood. In this study, we show that these cells are characterized by a molecular signature that mediates their retention in the liver. By examining liver transplants where donors and recipients are HLA mismatched, we distinguish between donor liver–derived and recipient-derived leukocytes to show that Eomeslo NK cells circulate freely whereas Eomeshi NK cells are unable to leave the liver. Furthermore, Eomeshi NK cells are retained in the liver for up to 13 y. Therefore, Eomeshi NK cells are long-lived liver-resident cells. We go on to show that Eomeshi NK cells can be recruited from the circulation during adult life and that circulating Eomeslo NK cells are able to upregulate Eomes and molecules mediating liver retention under cytokine conditions similar to those in the liver. This suggests that circulating NK cells are a precursor of their liver-resident counterparts. PMID:27798170

Role of natural killer cells in antibacterial immunity.

PubMed

Schmidt, Stanislaw; Ullrich, Evelyn; Bochennek, Konrad; Zimmermann, Stefanie-Yvonne; Lehrnbecher, Thomas

2016-12-01

Bacteria are a significant cause of infectious complications, in particular in immunocompromised patients. There is an increasing understanding that Natural Killer (NK) cells not only exhibit direct activity against bacteria, but also exert indirect antibacterial activity through interaction with other immune cells via cytokines and interferons. Areas covered: This review seeks to give a global overview of in vitro and in vivo data how NK cells interact with bacteria. In this regard, the review describes how NK cells directly damage and kill bacteria by soluble factors such as perforin, the impact of NK cells on other arms of the immune system, as well as how bacteria may inhibit NK cell activities. Expert commentary: A better characterization of the antibacterial effects of NK cells is urgently needed. With a better understanding of the interaction of NK cells and bacteria, NK cells may become a promising tool to prevent or to combat bacterial infections, e.g. by adoptively transferring NK cells to immunocompromised patients.

Recycling and resensitization of the neurokinin 1 receptor. Influence of agonist concentration and Rab GTPases.

PubMed

Roosterman, Dirk; Cottrell, Graeme S; Schmidlin, Fabien; Steinhoff, Martin; Bunnett, Nigel W

2004-07-16

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.

Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity.

PubMed

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.

CX3CR1-dependent recruitment of mature NK cells into the central nervous system contributes to control autoimmune neuroinflammation.

PubMed

Hertwig, Laura; Hamann, Isabell; Romero-Suarez, Silvina; Millward, Jason M; Pietrek, Rebekka; Chanvillard, Coralie; Stuis, Hanna; Pollok, Karolin; Ransohoff, Richard M; Cardona, Astrid E; Infante-Duarte, Carmen

2016-08-01

Fractalkine receptor (CX3CR1)-deficient mice develop very severe experimental autoimmune encephalomyelitis (EAE), associated with impaired NK cell recruitment into the CNS. Yet, the precise implications of NK cells in autoimmune neuroinflammation remain elusive. Here, we investigated the pattern of NK cell mobilization and the contribution of CX3CR1 to NK cell dynamics in the EAE. We show that in both wild-type and CX3CR1-deficient EAE mice, NK cells are mobilized from the periphery and accumulate in the inflamed CNS. However, in CX3CR1-deficient mice, the infiltrated NK cells displayed an immature phenotype contrasting with the mature infiltrates in WT mice. This shift in the immature/mature CNS ratio contributes to EAE exacerbation in CX3CR1-deficient mice, since transfer of mature WT NK cells prior to immunization exerted a protective effect and normalized the CNS NK cell ratio. Moreover, mature CD11b(+) NK cells show higher degranulation in the presence of autoreactive 2D2 transgenic CD4(+) T cells and kill these autoreactive cells more efficiently than the immature CD11b(-) fraction. Together, these data suggest a protective role of mature NK cells in EAE, possibly through direct modulation of T cells inside the CNS, and demonstrate that mature and immature NK cells are recruited into the CNS by distinct chemotactic signals. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Markers of nonselective and specific NK cell activation.

PubMed

Fogel, Leslie A; Sun, Michel M; Geurs, Theresa L; Carayannopoulos, Leonidas N; French, Anthony R

2013-06-15

NK cell activation is controlled by the integration of signals from cytokine receptors and germline-encoded activation and inhibitory receptors. NK cells undergo two distinct phases of activation during murine CMV (MCMV) infection: a nonselective phase mediated by proinflammatory cytokines and a specific phase driven by signaling through Ly49H, an NK cell activation receptor that recognizes infected cells. We sought to delineate cell surface markers that could distinguish NK cells that had been activated nonselectively from those that had been specifically activated through NK cell receptors. We demonstrated that stem cell Ag 1 (Sca-1) is highly upregulated during viral infections (to an even greater extent than CD69) and serves as a novel marker of early, nonselective NK cell activation. Indeed, a greater proportion of Sca-1(+) NK cells produced IFN-γ compared with Sca-1(-) NK cells during MCMV infection. In contrast to the universal upregulation of Sca-1 (as well as KLRG1) on NK cells early during MCMV infection, differential expression of Sca-1, as well as CD27 and KLRG1, was observed on Ly49H(+) and Ly49H(-) NK cells late during MCMV infection. Persistently elevated levels of KLRG1 in the context of downregulation of Sca-1 and CD27 were observed on NK cells that expressed Ly49H. Furthermore, the differential expression patterns of these cell surface markers were dependent on Ly49H recognition of its ligand and did not occur solely as a result of cellular proliferation. These findings demonstrate that a combination of Sca-1, CD27, and KLRG1 can distinguish NK cells nonselectively activated by cytokines from those specifically stimulated through activation receptors.

TGF-β1 Downregulates the Expression of CX3CR1 by Inducing miR-27a-5p in Primary Human NK Cells

PubMed Central

Regis, Stefano; Caliendo, Fabio; Dondero, Alessandra; Casu, Beatrice; Romano, Filomena; Loiacono, Fabrizio; Moretta, Alessandro; Bottino, Cristina; Castriconi, Roberta

2017-01-01

Activity of human natural killer (NK) cells against cancer cells is deeply suppressed by TGF-β1, an immunomodulatory cytokine that is released and activated in the tumor microenvironment. Moreover, our previous data showed that TGF-β1 modifies the chemokine receptor repertoire of NK cells. In particular, it decreases the expression of CX3CR1 that drives these effectors toward peripheral tissues, including tumor sites. To identify possible mechanisms mediating chemokine receptors modulation, we analyzed the microRNA profile of TGF-β1-treated primary NK cells. The analysis pointed out miR-27a-5p as a possible modulator of CX3CR1. We demonstrated the functional interaction of miR-27a-5p with the 3′ untranslated region (3′UTR) of CX3CR1 mRNA by two different experimental approaches: by the use of a luciferase assay based on a reporter construct containing the CX3CR1 3′UTR and by transfection of primary NK cells with a miR-27a-5p inhibitor. We also showed that the TGF-β1-mediated increase of miR-27a-5p expression is a consequence of miR-23a-27a-24-2 cluster induction. Moreover, we demonstrated that miR-27a-5p downregulates the surface expression of CX3CR1. Finally, we showed that neuroblastoma cells induced in resting NK cells a downregulation of the CX3CR1 expression that was paralleled by a significant increase of miR-27a-5p expression. Therefore, the present study highlights miR-27a-5p as a pivotal TGF-β1-induced regulator of CX3CR1 expression. PMID:28791023

Neurokinin B Exerts Direct Effects on the Ovary to Stimulate Estradiol Production.

PubMed

Qi, Xin; Salem, Mohamed; Zhou, Wenyi; Sato-Shimizu, Miwa; Ye, Gang; Smitz, Johan; Peng, Chun

2016-09-01

Neurokinin B (NKB) and its receptor, NK3R, play critical roles in reproduction by regulating the secretion of the hypothalamic GnRH. NKB and NK3R genes are also expressed in the ovary; however, their physiological roles within the ovary are unknown. The aim of this study was to determine whether NKB acts directly on the ovary to regulate reproduction. Injection of NKB into zebrafish accelerated follicle development, increased the mRNA levels of cyp11a1 and cyp19a1, and enhanced estradiol production. Similarly, NKB induced cyp11a1 and cyp19a1 expression in primary cultures of zebrafish follicular cells and stimulated estradiol production from cultured follicles. Furthermore, NKB activates cAMP response element-binding protein and ERK, and ERK inhibitors abolished the effect of NKB on cyp11a1, whereas protein kinase A and calmodulin-dependent protein kinase II inhibitors that blocked the activation of cAMP response element-binding protein, attenuated the effect of NKB on cyp19a1 expression. In a human granulosa cell line, COV434, a NKB agonist, senktide, also increased CYP11A1 and CYP19A1 mRNA levels and enhanced aromatase protein levels and activities. Small interfering RNA-mediated knockdown of NK3R reduced senktide-induced CYP11A1 and CYP19A1 mRNA levels. Finally, we found that NK3R mRNA was strongly down-regulated in granulosa cells obtained from polycystic ovary syndrome (PCOS) patients when compared with non-PCOS subjects. Taken together, our findings establish a direct action of NKB to induce ovarian estrogen production and raise the possibility that defective signaling of this pathway may contribute to the development of PCOS.

Intracellular staining for analysis of the expression and phosphorylation of signal transducers and activators of transcription (STATs) in NK cells.

PubMed

Miyagi, Takuya; Lee, Seung-Hwan; Biron, Christine A

2010-01-01

Cytokines stimulate biological responses by activating intracellular signaling pathways. We have been adapting flow cytometric techniques to measure the levels of expression and activation of signaling molecules within mixed populations containing NK cells and to characterize their differences within NK cell subpopulations. Approaches for evaluating the total levels of the signal transducers and activators of transcription STAT1 and STAT4, of STAT1 in cells expressing IFNgamma, and of the type 1 interferon (type 1 IFN) activation by phosphorylation, i.e., induction of pSTAT1 and pSTAT4, have been developed. The results of experiments using these techniques have demonstrated that an unusual feature of NK cells is high basal expression of STAT4 but reduced STAT1 levels. The condition predisposes for pSTAT4 activation by type 1 IFNs. The work has also shown, however, that total STAT1 levels are induced during viral infections as a result of IFN exposure, and that this change acts to promote the activation of STAT1 but limit both the activation of STAT4 and IFNgamma expression. The intracellular staining approaches used for the studies described here have utility in characterizing other mechanisms regulating cytokine-mediated signaling, and defining additional pathways shaping cellular responses to cytokines.

IL-15 super-agonist (ALT-803) enhances natural killer (NK) cell function against ovarian cancer

PubMed Central

Felices, M.; Chu, S.; Kodal, B.; Bendzick, L.; Ryan, C.; Lenvik, A.J.; Boylan, K.L.M.; Wong, H.C.; Skubitz, A.P.N.; Miller, J.S.; Geller, M.A.

2017-01-01

Objective Natural killer (NK) cells represent a powerful immunotherapeutic target as they lyse tumors directly, do not require differentiation, and can elicit potent inflammatory responses. The objective of these studies was to use an IL-15 super-agonist complex, ALT-803 (Altor BioScience Corporation), to enhance the function of both normal and ovarian cancer patient derived NK cells by increasing cytotoxicity and cytokine production. Methods NK cell function from normal donor peripheral blood mononuclear cells (PBMCs) and ovarian cancer patient ascites was assessed using flow cytometry and chromium release assays +/− ALT-803 stimulation. To evaluate the ability of ALT-803 to enhance NK cell function in vivo against ovarian cancer, we used a MA148-luc ovarian cancer NOD scid gamma (NSG) xenogeneic mouse model with transferred human NK cells. Results ALT-803 potently enhanced functionality of NK cells against all ovarian cancer cell lines with significant increases seen in CD107a, IFNγ and TNFα expression depending on target cell line. Function was also rescued in NK cells derived from ovarian cancer patient ascites. Finally, only animals treated with intraperitoneal ALT-803 displayed an NK dependent significant decrease in tumor. Conclusions ALT-803 enhances NK cell cytotoxicity against ovarian cancer in vitro and in vivo and is able to rescue functionality of NK cells derived from ovarian cancer patient ascites. These findings suggest that ALT-803 has the potential to enhance NK-cell-based immunotherapeutic approaches for the treatment of ovarian cancer. PMID:28236454

CD11c identifies a subset of murine liver natural killer cells that responds to adenoviral hepatitis

PubMed Central

Burt, Bryan M.; Plitas, George; Stableford, Jennifer A.; Nguyen, Hoang M.; Bamboat, Zubin M.; Pillarisetty, Venu G.; DeMatteo, Ronald P.

2008-01-01

The liver contains a unique repertoire of immune cells and a particular abundance of NK cells. We have found that CD11c defines a distinct subset of NK cells (NK1.1+CD3−) in the murine liver whose function was currently unknown. In naïve animals, CD11c+ liver NK cells displayed an activated phenotype and possessed enhanced effector functions when compared with CD11c− liver NK cells. During the innate response to adenovirus infection, CD11c+ NK cells were the more common IFN-γ-producing NK cells in the liver, demonstrated enhanced lytic capability, and gained a modest degree of APC function. The mechanism of IFN-γ production in vivo depended on TLR9 ligation as well as IL-12 and -18. Taken together, our findings demonstrate that CD11c+ NK cells are a unique subset of NK cells in the murine liver that contribute to the defense against adenoviral hepatitis. PMID:18664530

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

Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells.

PubMed

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A; Multhoff, Gabriele

2005-06-15

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

Heat Shock Protein 70 Surface-Positive Tumor Exosomes Stimulate Migratory and Cytolytic Activity of Natural Killer Cells

PubMed Central

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A.; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A.

2006-01-01

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo−/Colo+) and colon (CX−/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo−/CX exosomes were Hsp70 membrane derived positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes. PMID:15958569

Ex vivo expansion of highly cytotoxic human NK cells by cocultivation with irradiated tumor cells for adoptive immunotherapy.

PubMed

Lim, Seon Ah; Kim, Tae-Jin; Lee, Jung Eun; Sonn, Chung Hee; Kim, Kwanghee; Kim, Jiyoung; Choi, Jong Gwon; Choi, Il-Kyu; Yun, Chae-Ok; Kim, Jae-Hong; Yee, Cassian; Kumar, Vinay; Lee, Kyung-Mi

2013-04-15

Adoptive natural killer (NK) cell therapy may offer an effective treatment regimen for cancer patients whose disease is refractory to conventional therapy. NK cells can kill a wide range of tumor cells by patterned recognition of target ligands. We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the basis for developing a novel ex vivo expansion process. By screening class I-negative or -mismatched tumor cell lines we identified a Jurkat T-lymphoblast subline termed KL-1, which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity, accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of activation receptors, natural cytotoxicity receptors (NKp30, NKp44), and adhesion molecules (CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo models. KL-1-mediated expansion of NK cells was contact dependent and required interactions with CD16, the Fcγ receptor on NK cells, with ligands that are expressed on B cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while addition of EBV-transformed B cells further augmented NK expansion to approximately 740-fold. Together, our studies define a novel method for efficient activation of human NK cells that employs KL-1-lysed tumor cells and cocultured B cells, which drive a robust expansion of potent antitumor effector cells that will be useful for clinical evaluation. ©2012 AACR.

A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity employing NK or high-affinity NK cells in combination with cetuximab.

PubMed

Fujii, Rika; Schlom, Jeffrey; Hodge, James W

2018-05-01

OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma. METHODS Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells have been shown to influence the degree of ADCC of tumor cells, with the high-affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high-affinity CD16 allele. These irradiated high-affinity (ha)NK cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles. RESULTS Here the authors demonstrate for the first time 1) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; 2) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; 3) that engineered haNK cells-that is, cells transduced to express the CD16 V158 FcγRIIIa receptor-bind cetuximab with similar affinity to normal NK cells expressing the high-affinity VV allele; and 4) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. CONCLUSIONS These studies provide rationale for the use of cetuximab in combination with irradiated haNK cells for therapy for chordoma.

The influence of macrophages and the tumor microenvironment on natural killer cells.

PubMed

Krneta, T; Gillgrass, A; Ashkar, A A

2013-01-01

Numerous reviews in the field of NK cell biology dictate the pivotal role that NK cells play in tumor rejection. Although these cell types were originally described based on their cytotoxic ability, we now know that NK cells are not naturally born to kill. Both cellular interactions and the local environment in which the NK cell resides in may influence its cytotoxic functions. Just as organ specific NK cells have distinct phenotypic and functional differences, the tumor is a unique microenvironment in itself. The NK cells originally recruited to the tumor site are able to stimulate immune responses and aid in tumor destruction but eventually become persuaded otherwise by mechanisms of immunosuppression. Here, we review potential mechanisms and players involved in NK cell immunosuppression. In particular the effects of another innate immune player, macrophages, will be addressed in augmenting immunosuppression of NK cells within tumors. Tumor-associated macrophages (TAMs) are the main regulatory population of myeloid cells in the tumor and are characterized by their ability to promote tumor cell proliferation and metastasis. In addition, they express/release immunoregulatory factors which have been shown to directly inhibit NK cell function. Understanding how these two cell types interact in the distinct tumor microenvironment will allow us to consider therapies that target TAMs to promote enhanced NK cell activity.

CD94 Defines Phenotypically and Functionally Distinct Mouse NK Cell Subsets1

PubMed Central

Yu, Jianhua; Wei, Min; Mao, Hsiaoyin; Zhang, Jianying; Hughes, Tiffany; Mitsui, Takeki; Park, Il-kyoo; Hwang, Christine; Liu, Shujun; Marcucci, Guido; Trotta, Rossana; Benson, Don M.; Caligiuri, Michael A.

2010-01-01

Understanding of heterogeneous NK subsets is important for the study of NK cell biology and development, and for the application of NK cell-based therapies in the treatment of disease. Here we demonstrate that the surface expression of CD94 can distinctively divide mouse NK cells into two approximately even CD94low and CD94high subsets in all tested organs and tissues. The CD94high NK subset has significantly greater capacity to proliferate, produce IFN-γ, and lyse target cells than does the CD94low subset. The CD94high subset has exclusive expression of NKG2A/C/E, higher expression of CD117 and CD69, and lower expression of Ly49D (activating) and Ly49G2 (inhibitory). In vivo, purified mouse CD94low NK cells become CD94high NK cells, but not vice versa. Collectively, our data suggest that CD94 is an Ag that can be used to identify functionally distinct NK cell subsets in mice and could also be relevant to late-stage mouse NK cell development. PMID:19801519

Interferon-γ production by tubulointerstitial human CD56bright natural killer cells contributes to renal fibrosis and chronic kidney disease progression.

PubMed

Law, Becker M P; Wilkinson, Ray; Wang, Xiangju; Kildey, Katrina; Lindner, Mae; Rist, Melissa J; Beagley, Kenneth; Healy, Helen; Kassianos, Andrew J

2017-07-01

Natural killer (NK) cells are a population of lymphoid cells that play a significant role in mediating innate immune responses. Studies in mice suggest a pathological role for NK cells in models of kidney disease. In this study, we characterized the NK cell subsets present in native kidneys of patients with tubulointerstitial fibrosis, the pathological hallmark of chronic kidney disease. Significantly higher numbers of total NK cells (CD3 - CD56 + ) were detected in renal biopsies with tubulointerstitial fibrosis compared with diseased biopsies without fibrosis and healthy kidney tissue using multi-color flow cytometry. At a subset level, both the CD56 dim NK cell subset and particularly the CD56 bright NK cell subset were elevated in fibrotic kidney tissue. However, only CD56 bright NK cells significantly correlated with the loss of kidney function. Expression of the tissue-retention and -activation molecule CD69 on CD56 bright NK cells was significantly increased in fibrotic biopsy specimens compared with non-fibrotic kidney tissue, indicative of a pathogenic phenotype. Further flow cytometric phenotyping revealed selective co-expression of activating receptor CD335 (NKp46) and differentiation marker CD117 (c-kit) on CD56 bright NK cells. Multi-color immunofluorescent staining of fibrotic kidney tissue localized the accumulation of NK cells within the tubulointerstitium, with CD56 bright NK cells (NKp46 + CD117 + ) identified as the source of pro-inflammatory cytokine interferon-γ within the NK cell compartment. Thus, activated interferon-γ-producing CD56 bright NK cells are positioned to play a key role in the fibrotic process and progression to chronic kidney disease. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

The Broad Spectrum of Human Natural Killer Cell Diversity.

PubMed

Freud, Aharon G; Mundy-Bosse, Bethany L; Yu, Jianhua; Caligiuri, Michael A

2017-11-21

Natural killer (NK) cells provide protection against infectious pathogens and cancer. For decades it has been appreciated that two major NK cell subsets (CD56 bright and CD56 dim ) exist in humans and have distinct anatomical localization patterns, phenotypes, and functions in immunity. In light of this traditional NK cell dichotomy, it is now clear that the spectrum of human NK cell diversity is much broader than originally appreciated as a result of variegated surface receptor, intracellular signaling molecule, and transcription factor expression; tissue-specific imprinting; and foreign antigen exposure. The recent discoveries of tissue-resident NK cell developmental intermediates, non-NK innate lymphoid cells, and the capacity for NK cells to adapt and differentiate into long-lived memory cells has added further complexity to this field. Here we review our current understanding of the breadth and generation of human NK cell diversity. Copyright © 2017 Elsevier Inc. All rights reserved.

Chimeric antigen receptor (CAR)-modified natural killer cell-based immunotherapy and immunological synapse formation in cancer and HIV.

PubMed

Liu, Dongfang; Tian, Shuo; Zhang, Kai; Xiong, Wei; Lubaki, Ndongala Michel; Chen, Zhiying; Han, Weidong

2017-12-01

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells contribute to the body's immune defenses. Current chimeric antigen receptor (CAR)-modified T cell immunotherapy shows strong promise for treating various cancers and infectious diseases. Although CAR-modified NK cell immunotherapy is rapidly gaining attention, its clinical applications are mainly focused on preclinical investigations using the NK92 cell line. Despite recent advances in CAR-modified T cell immunotherapy, cost and severe toxicity have hindered its widespread use. To alleviate these disadvantages of CAR-modified T cell immunotherapy, additional cytotoxic cell-mediated immunotherapies are urgently needed. The unique biology of NK cells allows them to serve as a safe, effective, alternative immunotherapeutic strategy to CAR-modified T cells in the clinic. While the fundamental mechanisms underlying the cytotoxicity and side effects of CAR-modified T and NK cell immunotherapies remain poorly understood, the formation of the immunological synapse (IS) between CAR-modified T or NK cells and their susceptible target cells is known to be essential. The role of the IS in CAR T and NK cell immunotherapies will allow scientists to harness the power of CAR-modified T and NK cells to treat cancer and infectious diseases. In this review, we highlight the potential applications of CAR-modified NK cells to treat cancer and human immunodeficiency virus (HIV), and discuss the challenges and possible future directions of CAR-modified NK cell immunotherapy, as well as the importance of understanding the molecular mechanisms of CAR-modified T cell- or NK cell-mediated cytotoxicity and side effects, with a focus on the CAR-modified NK cell IS.

A Batf3/Nlrp3/IL-18 Axis Promotes Natural Killer Cell IL-10 Production during Listeria monocytogenes Infection.

PubMed

Clark, Sarah E; Schmidt, Rebecca L; McDermott, Daniel S; Lenz, Laurel L

2018-05-29

The bacterial pathogen Listeria monocytogenes (Lm) capitalizes on natural killer (NK) cell production of regulatory interleukin (IL)-10 to establish severe systemic infections. Here, we identify regulators of this IL-10 secretion. We show that IL-18 signals to NK cells license their ability to produce IL-10. IL-18 acts independent of IL-12 and STAT4, which co-stimulate IFNγ secretion. Dendritic cell (DC) expression of Nlrp3 is required for IL-18 release in response to the Lm p60 virulence protein. Therefore, mice lacking Nlrp3, Il18, or Il18R fail to accumulate serum IL-10 and are highly resistant to systemic Lm infection. We further show that cells expressing or dependent on Batf3 are required for IL-18-inducing IL-10 production observed in infected mice. These findings explain how Il18 and Batf3 promote susceptibility to bacterial infection and demonstrate the ability of Lm to exploit NLRP3 for the promotion of regulatory NK cell activity. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Human NK cells: From surface receptors to clinical applications.

PubMed

Moretta, Lorenzo; Pietra, Gabriella; Vacca, Paola; Pende, Daniela; Moretta, Francesca; Bertaina, Alice; Mingari, Maria Cristina; Locatelli, Franco; Moretta, Alessandro

2016-10-01

Natural killer (NK) cells play a major role in innate defenses against pathogens, primarily viruses, and are also thought to be part of the immunosurveillance against tumors. They express an array of surface receptors that mediate NK cell function. The human leukocytes antigen (HLA) class I-specific inhibitory receptors allow NK cells to detect and kill cells that have lost or under-express HLA class I antigens, a typical feature of tumor or virally infected cells. However, NK cell activation and induction of cytolytic activity and cytokine production depends on another important checkpoint, namely the expression on target cells of ligands recognized by activating NK receptors. Despite their potent cytolytic activity, NK cells frequently fail to eliminate tumors. This is due to mechanisms of tumor escape, determined by the tumor cells themselves or by tumor-associated cells (i.e. the tumor microenvironment) via the release of soluble suppressive factors or the induction of inhibitory loops involving induction of regulatory T cells, M2-polarized macrophages and myeloid-derived suppressor cells. The most important clinical application involving NK cells is the cure of high-risk leukemias in the haplo-identical hematopoietic stem cell transplant (HSCT) setting. NK cells originated from hematopoietic stem cells (HSC) of HLA-haploidentical donors may express Killer Immunoglobulin-like receptors (KIRs) that are mismatched with the HLA class I alleles of the recipient. This allows NK cells to kill leukemia blasts residual after the conditioning regimen, while sparing normal cells (that do not express ligands for activating NK receptors). More recent approaches based on the specific removal of TCR α/β(+) T cells and of CD19(+) B cells, allow the infusion, together with CD34(+) HSC, of mature KIR(+) NK cells and of TCR γ/δ(+) T cells, both characterized by a potent anti-leukemia activity. This greatly reduces the time interval necessary to obtain alloreactive, KIR(+) NK cells derived from donor HSC. Another promising approach is based on the use of anti-KIR blocking monoclonal antibodies (mAbs), rendering alloreactive any KIR(+) NK cells. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Restoring Natural Killer Cell Immunity against Multiple Myeloma in the Era of New Drugs

PubMed Central

Pittari, Gianfranco; Vago, Luca; Festuccia, Moreno; Bonini, Chiara; Mudawi, Deena; Giaccone, Luisa; Bruno, Benedetto

2017-01-01

Transformed plasma cells in multiple myeloma (MM) are susceptible to natural killer (NK) cell-mediated killing via engagement of tumor ligands for NK activating receptors or “missing-self” recognition. Similar to other cancers, MM targets may elude NK cell immunosurveillance by reprogramming tumor microenvironment and editing cell surface antigen repertoire. Along disease continuum, these effects collectively result in a progressive decline of NK cell immunity, a phenomenon increasingly recognized as a critical determinant of MM progression. In recent years, unprecedented efforts in drug development and experimental research have brought about emergence of novel therapeutic interventions with the potential to override MM-induced NK cell immunosuppression. These NK-cell enhancing treatment strategies may be identified in two major groups: (1) immunomodulatory biologics and small molecules, namely, immune checkpoint inhibitors, therapeutic antibodies, lenalidomide, and indoleamine 2,3-dioxygenase inhibitors and (2) NK cell therapy, namely, adoptive transfer of unmanipulated and chimeric antigen receptor-engineered NK cells. Here, we summarize the mechanisms responsible for NK cell functional suppression in the context of cancer and, specifically, myeloma. Subsequently, contemporary strategies potentially able to reverse NK dysfunction in MM are discussed. PMID:29163516

The natural killer cell response to West Nile virus in young and old individuals with or without a prior history of infection.

PubMed

Yao, Yi; Strauss-Albee, Dara M; Zhou, Julian Q; Malawista, Anna; Garcia, Melissa N; Murray, Kristy O; Blish, Catherine A; Montgomery, Ruth R

2017-01-01

West Nile virus (WNV) typically leads to asymptomatic infection but can cause severe neuroinvasive disease or death, particularly in the elderly. Innate NK cells play a critical role in antiviral defenses, yet their role in human WNV infection is poorly defined. Here we demonstrate that NK cells mount a robust, polyfunctional response to WNV characterized by cytolytic activity, cytokine and chemokine secretion. This is associated with downregulation of activating NK cell receptors and upregulation of NK cell activating ligands for NKG2D. The NK cell response did not differ between young and old WNV-naïve subjects, but a history of symptomatic infection is associated with more IFN-γ producing NK cell subsets and a significant decline in a specific NK cell subset. This NK repertoire skewing could either contribute to or follow heightened immune pathogenesis from WNV infection, and suggests that NK cells could play an important role in WNV infection in humans.

The Rise of Allogeneic Natural Killer Cells As a Platform for Cancer Immunotherapy: Recent Innovations and Future Developments

PubMed Central

Veluchamy, John P.; Kok, Nina; van der Vliet, Hans J.; Verheul, Henk M. W.; de Gruijl, Tanja D.; Spanholtz, Jan

2017-01-01

Natural killer (NK) cells are critical immune effector cells in the fight against cancer. As NK cells in cancer patients are highly dysfunctional and reduced in number, adoptive transfer of large numbers of cytolytic NK cells and their potential to induce relevant antitumor responses are widely explored in cancer immunotherapy. Early studies from autologous NK cells have failed to demonstrate significant clinical benefit. In this review, the clinical benefits of adoptively transferred allogeneic NK cells in a transplant and non-transplant setting are compared and discussed in the context of relevant NK cell platforms that are being developed and optimized by various biotech industries with a special focus on augmenting NK cell functions. PMID:28620386

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. Copyright © 2015 John Wiley & Sons, Inc.

Decreased natural killer cell activity in atopic eczema.

PubMed Central

Hall, T J; Rycroft, R; Brostoff, J

1985-01-01

We have studied NK cell activity in atopic and non-atopic subjects using a standard 51Cr-release assay and K562 target cells. In atopics (AT) with allergic rhinitis and/or asthma, NK cell activity was similar to that in non-atopic (N) subjects, whilst patients with severe atopic eczema (AE) had depressed NK cell activity compared to AT or N subjects. In addition, circulating T-cell numbers and Con A responsiveness was decreased in AE, although neither parameter was correlated with decreased NK cell activity. However, decreased NK cell activity in atopic eczema was positively correlated with decreased numbers of Fc gamma + lymphocytes (P = 0.01) and decreased effector: target cell binding (P = 0.05), and negatively correlated with increased monocytes in AE (P = 0.09). AE NK cell activity was equally or more sensitive to the inhibitory effects of drugs such as dibutyryl cyclic AMP, prostaglandins (PG) D2,E2 and histamine. The relative percentage increase in NK cell activity by the interferon inducer poly I:C was similar in AE patients and controls. The results suggest that reduced numbers of circulating NK cells and pre-NK cells account for the depressed level of NK cell activity in subjects with severe atopic eczema. PMID:3876984

NK cell-released exosomes: Natural nanobullets against tumors.

PubMed

Fais, Stefano

2013-01-01

We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool.

NK cell-released exosomes

PubMed Central

Fais, Stefano

2013-01-01

We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool. PMID:23482694

Slp-76 is a critical determinant of NK-cell mediated recognition of missing-self targets.

PubMed

Lampe, Kristin; Endale, Mehari; Cashman, Siobhan; Fang, Hao; Mattner, Jochen; Hildeman, David; Hoebe, Kasper

2015-07-01

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying "missing-self" recognition, including the involvement of activating receptors, remain poorly understood. Using ethyl-N-nitrosourea mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell mediated recognition and elimination of "missing-self" targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation (Thr428Ile) in the SH2 domain of Slp-76-a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele-while no major defects were observed in conventional T-cell development/function, a marked defect in NK cell mediated elimination of β2-microglobulin (β2M) deficient target cells was observed. Further studies revealed Slp-76 to control NK-cell receptor expression and maturation; however, activation of Slp-76(ace/ace) NK cells through ITAM-containing NK-cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-β2M(-/-) target cell synapse revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76(ace/ace) NK cells. Overall these studies establish Slp-76 as a critical determinant of NK-cell development and NK cell mediated elimination of missing-self target cells in mice. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Slp-76 is a critical determinant of NK cell-mediated recognition of missing-self targets

PubMed Central

Lampe, Kristin; Endale, Mehari; Cashman, Siobhan; Fang, Hao; Mattner, Jochen; Hildeman, David; Hoebe, Kasper

2015-01-01

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying “missing-self” recognition, including the involvement of activating receptors, remain poorly understood. Using ENU mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell-mediated recognition and elimination of “missing-self” targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation [Thr428Ile] in the SH2 domain of Slp-76—a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele—while no major defects were observed in conventional T cell development/function, a marked defect in NK cell-mediated elimination of β2-Microglobulin (β2M)-deficient target cells was observed. Further studies revealed Slp-76 to control NK cell receptor expression and maturation, however, activation of Slp-76ace/ace NK cells through ITAM-containing NK cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-β2M−/− target cell synapse, revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76ace/ace NK cells. Overall these studies establish Slp-76 as a critical determinant of NK cell development and NK cell-mediated elimination of missing-self target cells. PMID:25929249

TLR-Stimulated Eosinophils Mediate Recruitment and Activation of NK Cells In Vivo.

PubMed

O'Flaherty, S M; Sutummaporn, K; Häggtoft, W L; Worrall, A P; Rizzo, M; Braniste, V; Höglund, P; Kadri, N; Chambers, B J

2017-06-01

Eosinophils like many myeloid innate immune cells can provide cytokines and chemokines for the activation of other immune cells upon TLR stimulation. When TLR-stimulated eosinophils were inoculated i.p. into wild-type mice, and NK cells were rapidly recruited and exhibited antitumour cytotoxicity. However, when mice depleted of CD11c + cells were used, a marked decrease in the number of recruited NK cells was observed. We postulated that CpG or LPS from the injected eosinophils could be transferred to host cells, which in turn could recruit NK cells. However, by inoculating mice deficient in TLR4 or TLR9 with LPS or CpG-stimulated eosinophils respectively, NK cell recruitment was still observed alongside cytotoxicity and IFNγ production. CpG stimulation of eosinophils produced the pro-inflammatory cytokine IL-12 and the chemokine CXCL10, which are important for NK cell activation and recruitment in vivo. To demonstrate the importance of CXCL10 in NK cell recruitment, we found that CpG-stimulated eosinophils pretreated with the gut microbial metabolite butyrate had reduced expression and production of CXCL10 and IL-12 and concomitantly were poor at recruitment of NK cells and inducing IFNγ in NK cells. Therefore, eosinophils like other innate immune cells of myeloid origin can conceivably stimulate NK cell activity. In addition, products of the gut microbiota can be potential inhibitors of NK cell. © 2017 The Foundation for the Scandinavian Journal of Immunology.

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

Substance P increases liver fibrosis by differential changes in senescence of cholangiocytes and hepatic stellate cells.

PubMed

Wan, Ying; Meng, Fanyin; Wu, Nan; Zhou, Tianhao; Venter, Julie; Francis, Heather; Kennedy, Lindsey; Glaser, Trenton; Bernuzzi, Francesca; Invernizzi, Pietro; Glaser, Shannon; Huang, Qiaobing; Alpini, Gianfranco

2017-08-01

Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct-ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin-1 receptor (NK-1R). To identify whether SP regulates liver fibrosis during cholestasis, wild-type or NK-1R knockout (NK-1R -/- ) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2 -/- ) mice treated with either an NK-1R antagonist (L-733,060) or saline were used. Additionally, wild-type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK-1R in both BDL and Mdr2 -/- mice compared to wild-type mice. Expression of tachykinin precursor 1 and NK-1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK-1R decreased BDL-induced liver fibrosis, and treatment with L-733,060 resulted in decreased liver fibrosis in Mdr2 -/- mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor-β1 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK-1R -/- mice with BDL surgery or Mdr2 -/- mice treated with L-733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L-733,060 inhibited SP-induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L-733,060 partially reversed the SP-induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP-induced increase of senescence-related gene expression in cultured cholangiocytes. Collectively, our results demonstrate the regulatory effects of the SP/NK-1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury. (Hepatology 2017;66:528-541). © 2017 by the American Association for the Study of Liver Diseases. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells.

PubMed

Sánchez-Martínez, Diego; Lanuza, Pilar M; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A; Pardo, Julián

2016-01-01

Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV ) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

PubMed

Klöß, Stephan; Oberschmidt, Olaf; Morgan, Michael; Dahlke, Julia; Arseniev, Lubomir; Huppert, Volker; Granzin, Markus; Gardlowski, Tanja; Matthies, Nadine; Soltenborn, Stephanie; Schambach, Axel; Koehl, Ulrike

2017-10-01

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56 + CD3 - ) was carried out with the CliniMACS Prodigy ® in a single process, starting with approximately 1.2 × 10 9 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10 6 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO ™ 10, CellGro ® , TexMACS ™ , and NK MACS ® ). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56 + CD3 - target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56 dim CD16 pos and CD56 bright CD16 dim&neg NK subsets on day 14 with cells cultivated in NK MACS ® media. Moreover, effector cell expansion in manually performed experiments with NK MACS ® containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123 pos AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

Confinement of activating receptors at the plasma membrane controls natural killer cell tolerance.

PubMed

Guia, Sophie; Jaeger, Baptiste N; Piatek, Stefan; Mailfert, Sébastien; Trombik, Tomasz; Fenis, Aurore; Chevrier, Nicolas; Walzer, Thierry; Kerdiles, Yann M; Marguet, Didier; Vivier, Eric; Ugolini, Sophie

2011-04-05

Natural killer (NK) cell tolerance to self is partly ensured by major histocompatibility complex (MHC) class I-specific inhibitory receptors on NK cells, which dampen their reactivity when engaged. However, NK cells that do not detect self MHC class I are not autoreactive. We used dynamic fluorescence correlation spectroscopy to show that MHC class I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC class I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We propose that the confinement of activating receptors at the plasma membrane is pivotal to ensuring the self-tolerance of NK cells.

Development and Function of CD94-Deficient Natural Killer Cells

PubMed Central

Orr, Mark T.; Wu, Jun; Fang, Min; Sigal, Luis J.; Spee, Pieter; Egebjerg, Thomas; Dissen, Erik; Fossum, Sigbjørn; Phillips, Joseph H.; Lanier, Lewis L.

2010-01-01

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions. PMID:21151939

Development and function of CD94-deficient natural killer cells.

PubMed

Orr, Mark T; Wu, Jun; Fang, Min; Sigal, Luis J; Spee, Pieter; Egebjerg, Thomas; Dissen, Erik; Fossum, Sigbjørn; Phillips, Joseph H; Lanier, Lewis L

2010-12-03

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions.

Cytokine-Induced Memory-Like Differentiation Enhances Unlicensed Natural Killer Cell Antileukemia and FcγRIIIa-Triggered Responses.

PubMed

Wagner, Julia A; Berrien-Elliott, Melissa M; Rosario, Maximillian; Leong, Jeffrey W; Jewell, Brea A; Schappe, Timothy; Abdel-Latif, Sara; Fehniger, Todd A

2017-03-01

Cytokine-induced memory-like natural killer (NK) cells differentiate after short-term preactivation with IL-12, IL-15, and IL-18 and display enhanced effector function in response to cytokines or tumor targets for weeks after the initial preactivation. Conventional NK cell function depends on a licensing signal, classically delivered by an inhibitory receptor engaging its cognate MHC class I ligand. How licensing status integrates with cytokine-induced memory-like NK cell responses is unknown. We investigated this interaction using killer cell immunoglobulin-like receptor- and HLA-genotyped primary human NK cells. Memory-like differentiation resulted in enhanced IFN-γ production triggered by leukemia targets or FcγRIIIa ligation within licensed NK cells, which exhibited the highest functionality of the NK cell subsets interrogated. IFN-γ production by unlicensed memory-like NK cells was also enhanced to a level comparable with that of licensed control NK cells. Mechanistically, differences in responses to FcγRIIIa-based triggering were not explained by alterations in key signaling intermediates, indicating that the underlying biology of memory-like NK cells is distinct from that of adaptive NK cells in human cytomegalovirus-positive individuals. Additionally, memory-like NK cells responded robustly to cytokine receptor restimulation with no impact of licensing status. These results demonstrate that both licensed and unlicensed memory-like NK cell populations have enhanced functionality, which may be translated to improve leukemia immunotherapy. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Human Natural Killer Cells Exhibit Direct Activity Against Aspergillus fumigatus Hyphae, But Not Against Resting Conidia

PubMed Central

Schmidt, Stanislaw; Tramsen, Lars; Hanisch, Mitra; Latgé, Jean-Paul; Huenecke, Sabine; Koehl, Ulrike

2011-01-01

Because natural killer (NK) cells kill tumor cells and combat infections, there is growing interest in adoptively transferring NK cells to hematopoietic stem cell recipients. Unfortunately, in humans, the activity of NK cells against Aspergillus species, the major cause of invasive fungal infection in stem cell recipients, are poorly characterized. Our results show that unstimulated and interleukin-2 prestimulated human NK cells kill Aspergillus fumigatus hyphae but do not affect resting conidia. Killing is also induced by the supernatant of prestimulated NK cells and human perforin. The high levels of interferon-γ and granulocyte macrophage colony-stimulating factor produced by prestimulated NK cells are significantly reduced by Aspergillus, indicating an immunosuppressive effect of the fungus. Whereas Aspergillus hyphae activate NK cells, resting, and germinating, conidia and conidia of ΔrodA mutants lacking the hydrophobic surface layer do not. Our results suggest that adoptively transferred human NK cells may be a potential antifungal tool in the transplantation context. PMID:21208932

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.

Natural killer (NK) cells inhibit systemic metastasis of glioblastoma cells and have therapeutic effects against glioblastomas in the brain.

PubMed

Lee, Se Jeong; Kang, Won Young; Yoon, Yeup; Jin, Ju Youn; Song, Hye Jin; Her, Jung Hyun; Kang, Sang Mi; Hwang, Yu Kyeong; Kang, Kyeong Jin; Joo, Kyeung Min; Nam, Do-Hyun

2015-12-24

Glioblastoma multiforme (GBM) is characterized by extensive local invasion, which is in contrast with extremely rare systemic metastasis of GBM. Molecular mechanisms inhibiting systemic metastasis of GBM would be a novel therapeutic candidate for GBM in the brain. Patient-derived GBM cells were primarily cultured from surgical samples of GBM patients and were inoculated into the brains of immune deficient BALB/c-nude or NOD-SCID IL2Rgamma(null) (NSG) mice. Human NK cells were isolated from peripheral blood mononucleated cells and expanded in vitro. Patient-derived GBM cells in the brains of NSG mice unexpectedly induced spontaneous lung metastasis although no metastasis was detected in BALB/c-nude mice. Based on the difference of the innate immunity between two mouse strains, NK cell activities of orthotopic GBM xenograft models based on BALB/c-nude mice were inhibited. NK cell inactivation induced spontaneous lung metastasis of GBM cells, which indicated that NK cells inhibit the systemic metastasis. In vitro cytotoxic activities of human NK cells against GBM cells indicated that cytotoxic activity of NK cells against GBM cells prevents systemic metastasis of GBM and that NK cells could be effective cell therapeutics against GBM. Accordingly, NK cells transplanted into orthotopic GBM xenograft models intravenously or intratumorally induced apoptosis of GBM cells in the brain and showed significant therapeutic effects. Our results suggest that innate NK immunity is responsible for rare systemic metastasis of GBM and that sufficient supplementation of NK cells could be a promising immunotherapeutic strategy for GBM in the brain.

Implication of Highly Cytotoxic Natural Killer Cells for Esophageal Squamous Cell Carcinoma Treatment.

PubMed

Lim, Kee Siang; Mimura, Kosaku; Kua, Ley-Fang; Shiraishi, Kensuke; Kono, Koji

2018-04-20

Esophageal squamous cell carcinoma (ESCC) is an aggressive upper gastrointestinal cancer and effective treatments are limited. Previous studies reported that natural killer (NK) cells expanded by coculturing with K562-mb15-41BBL feeder cells, a genetically modified K562 leukemia cell line that expresses membrane-bound interleukin (IL)-15 and 41BBL ligand, were highly proliferative and highly cytotoxic. Here, we investigated the potential of expanded NK cells for ESCC treatment. We analyzed both genetic and surface expression levels of NKG2D ligands (NKG2DLs) in ESCC using publicly available microarray data sets and ESCC cell lines. The cytotoxicity of resting and of IL-2-activated NK cells against ESCC cell lines was compared with that of expanded NK cells. We then also investigated the effect of epithelial mesenchymal transition (EMT) inducers, GSK3β inhibitor and epidermal growth factor, on NKG2DLs expressions. As a result, MICA and MICB were significantly overexpressed in ESCC compared with adjacent normal tissues and surface NKG2DLs were expressed in ESCC cell lines. Expanded NK cells were much potent than IL-2-activated and resting NK cells against ESCC cell lines. Blocking of NKG2D with anti-NKG2D monoclonal antibody dampened expanded NK cell cytotoxicity, suggesting that the NKG2DLs-NKG2D interaction is crucial for NK cells to eliminate ESCC cells. EMT inducers concurrently induced EMT and NKG2DLs expression in ESCC cell lines rendering transitioned cells more sensitive to expanded NK cells. In conclusion, expanded NK cells were highly cytotoxic against NKG2DLs-expressing ESCC cells, particularly the EMT phenotype. These results provide a strong rationale for clinical use of these NK cells in ESCC patients.

Mycobacterium tuberculosis infection modulates adipose tissue biology

PubMed Central

Kühl, Anja A.; Kupz, Andreas; Vogelzang, Alexis; Mollenkopf, Hans-Joachim; Löwe, Delia; Bandermann, Silke; Dorhoi, Anca; Brinkmann, Volker

2017-01-01

Mycobacterium tuberculosis (Mtb) primarily resides in the lung but can also persist in extrapulmonary sites. Macrophages are considered the prime cellular habitat in all tissues. Here we demonstrate that Mtb resides inside adipocytes of fat tissue where it expresses stress-related genes. Moreover, perigonadal fat of Mtb-infected mice disseminated the infection when transferred to uninfected animals. Adipose tissue harbors leukocytes in addition to adipocytes and other cell types and we observed that Mtb infection induces changes in adipose tissue biology depending on stage of infection. Mice infected via aerosol showed infiltration of inducible nitric oxide synthase (iNOS) or arginase 1 (Arg1)-negative F4/80+ cells, despite recruitment of CD3+, CD4+ and CD8+ T cells. Gene expression analysis of adipose tissue of aerosol Mtb-infected mice provided evidence for upregulated expression of genes associated with T cells and NK cells at 28 days post-infection. Strikingly, IFN-γ-producing NK cells and Mtb-specific CD8+ T cells were identified in perigonadal fat, specifically CD8+CD44-CD69+ and CD8+CD44-CD103+ subpopulations. Gene expression analysis of these cells revealed that they expressed IFN-γ and the lectin-like receptor Klrg1 and down-regulated CD27 and CD62L, consistent with an effector phenotype of Mtb-specific CD8+ T cells. Sorted NK cells expressed higher abundance of Klrg1 upon infection, as well. Our results reveal the ability of Mtb to persist in adipose tissue in a stressed state, and that NK cells and Mtb-specific CD8+ T cells infiltrate infected adipose tissue where they produce IFN-γ and assume an effector phenotype. We conclude that adipose tissue is a potential niche for Mtb and that due to infection CD8+ T cells and NK cells are attracted to this tissue. PMID:29040326

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish.

PubMed

Moore, Finola E; Garcia, Elaine G; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C; Garcia, Amaris J; Mylvaganam, Ravi; Yoder, Jeffrey A; Blackburn, Jessica S; Sadreyev, Ruslan I; Ceol, Craig J; North, Trista E; Langenau, David M

2016-05-30

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4(+) cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2(E450fs) mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4(+) cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2(E450fs) mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4(+)/CD8(+) cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. © 2016 Moore et al.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish

PubMed Central

Garcia, Elaine G.; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C.; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C.; Garcia, Amaris J.; Mylvaganam, Ravi; Yoder, Jeffrey A.; Blackburn, Jessica S.; Sadreyev, Ruslan I.; Ceol, Craig J.; North, Trista E.

2016-01-01

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4+ cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2E450fs mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4+ cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2E450fs mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4+/CD8+ cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb. In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. PMID:27139488

Boosting Natural Killer Cell-Based Immunotherapy with Anticancer Drugs: a Perspective.

PubMed

Cifaldi, Loredana; Locatelli, Franco; Marasco, Emiliano; Moretta, Lorenzo; Pistoia, Vito

2017-12-01

Natural killer (NK) cells efficiently recognize and kill tumor cells through several mechanisms including the expression of ligands for NK cell-activating receptors on target cells. Different clinical trials indicate that NK cell-based immunotherapy represents a promising antitumor treatment. However, tumors develop immune-evasion strategies, including downregulation of ligands for NK cell-activating receptors, that can negatively affect antitumor activity of NK cells, which either reside endogenously, or are adoptively transferred. Thus, restoration of the expression of NK cell-activating ligands on tumor cells represents a strategic therapeutic goal. As discussed here, various anticancer drugs can fulfill this task via different mechanisms. We envision that the combination of selected chemotherapeutic agents with NK cell adoptive transfer may represent a novel strategy for cancer immunotherapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Natural Killer Cells Promote Fetal Development through the Secretion of Growth-Promoting Factors.

PubMed

Fu, Binqing; Zhou, Yonggang; Ni, Xiang; Tong, Xianhong; Xu, Xiuxiu; Dong, Zhongjun; Sun, Rui; Tian, Zhigang; Wei, Haiming

2017-12-19

Natural killer (NK) cells are present in large populations at the maternal-fetal interface during early pregnancy. However, the role of NK cells in fetal growth is unclear. Here, we have identified a CD49a + Eomes + subset of NK cells that secreted growth-promoting factors (GPFs), including pleiotrophin and osteoglycin, in both humans and mice. The crosstalk between HLA-G and ILT2 served as a stimulus for GPF-secreting function of this NK cell subset. Decreases in this GPF-secreting NK cell subset impaired fetal development, resulting in fetal growth restriction. The transcription factor Nfil3, but not T-bet, affected the function and the number of this decidual NK cell subset. Adoptive transfer of induced CD49a + Eomes + NK cells reversed impaired fetal growth and rebuilt an appropriate local microenvironment. These findings reveal properties of NK cells in promoting fetal growth. In addition, this research proposes approaches for therapeutic administration of NK cells in order to reverse restricted nourishments within the uterine microenvironment during early pregnancy. Copyright © 2017 Elsevier Inc. All rights reserved.

Dynamic changes in the numbers of different subsets of peripheral blood NK cells in patients with systemic lupus erythematosus following classic therapy.

PubMed

Ma, Hongshuang; Zhao, Ling; Jiang, Zhenyu; Jiang, Yanfang; Feng, Li; Ye, Zhuang

2014-11-01

Imbalance of natural killer (NK) cells is associated with the development of systemic lupus erythematosus (SLE). However, little is known about the dynamic changes on NK cells following therapy. This study aimed at examining the impact of classic therapies on the numbers of different subsets of NK cells in new-onset SLE patients. The numbers of different subsets of peripheral blood NK cells in 24 new-onset SLE patients before, 4 and 12 weeks post the classic therapies, and 7 healthy controls were determined by flow cytometry. The potential correlation between the numbers of NK cells and the values of clinical measures was analyzed. In comparison with that before treatment, the numbers of NK, NKG2C+, and KIR2DL3+ NK cells were significantly increased while the numbers of NKp46+ and NKG2A + NK cells significantly decreased at 4 and/or 12 weeks post the treatment only in the drug well-responding patients, but not in those poor responders (P < 0.05 for all). The numbers of NKG2C + NK cells were correlated positively with the levels of serum C3 while the numbers of KIR2DL3+ NK cells were correlated negatively with the scores of SLEDAI in these patients at 4 weeks post the treatment. The classic therapies modulated the numbers of some subsets of NK cells in drug well-responding SLE patients. The changes in the numbers of some subsets of NK cells may serve as biomarkers for evaluating the therapeutic responses of SLE.

Characterization of tumor infiltrating Natural Killer cell subset

PubMed Central

Nissan, Aviram; Darash-Yahana, Merav; Peretz, Tamar; Mandelboim, Ofer; Rachmilewitz, Jacob

2015-01-01

The presence of tumor-infiltrating Natural Killer (NK) within a tumor bed may be indicative of an ongoing immune response toward the tumor. However, many studies have shown that an intense NK infiltration, is associated with advanced disease and may even facilitate cancer development. The exact role of the tumor infiltrating NK cells and the correlation between their presence and poor prognosis remains unclear. Interestingly, during pregnancy high numbers of a specific NK subset, CD56brightCD16dim, are accumulated within first trimester deciduas. These decidual NK (dNK) cells are unique in their gene expression pattern secret angiogenic factors that induce vascular growth. In the present study we demonstrate a significant enrichment of a CD56brighCD16dim NK cells within tumors. These NK cells express several dNK markers including VEGF. Hence, this study adds new insights into the identity of tumor residual NK cells, which has clear implications for the treatment of human cancer. PMID:26079948

Expression of activating natural killer-cell receptors is a hallmark of the innate-like T-cell neoplasm in peripheral T-cell lymphomas.

PubMed

Uemura, Yu; Isobe, Yasushi; Uchida, Akiko; Asano, Junko; Nishio, Yuji; Sakai, Hirotaka; Hoshikawa, Masahiro; Takagi, Masayuki; Nakamura, Naoya; Miura, Ikuo

2018-04-01

Peripheral T- or natural killer (NK)-cell lymphomas are rare and difficult-to-recognize diseases. It remains arduous to distinguish between NK cell- and cytotoxic T-lymphocyte-derived lymphomas through routine histological evaluation. To clarify the cells of origin, we focused on NK-cell receptors and examined the expression using immunohistochemistry in 22 cases with T- and NK-cell neoplasms comprising angioimmunoblastic T-cell lymphoma, anaplastic lymphoma kinase (ALK)-positive and -negative anaplastic large-cell lymphomas, extranodal NK/T-cell lymphoma, nasal type, monomorphic epitheliotropic intestinal T-cell lymphoma, aggressive NK-cell leukemia, and other peripheral T-cell lymphomas. Inhibitory receptor leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) was detected in 14 (64%) cases, whereas activating receptors DNAM1, NKp46, and NKG2D were expressed in 7 (32%), 9 (41%), and 5 (23%) cases, respectively. Although LILRB1 was detected regardless of the disease entity, the activating NK-cell receptors were expressed predominantly in TIA-1-positive neoplasms (DNAM1, 49%; NKp46, 69%; and NKG2D, 38%). In addition, NKp46 and NKG2D were detected only in NK-cell neoplasms and cytotoxic T-lymphocyte-derived lymphomas including monomorphic epitheliotropic intestinal T-cell lymphoma. One Epstein-Barr virus-harboring cytotoxic T-lymphocyte-derived lymphoma mimicking extranodal NK/T-cell lymphoma, nasal type lacked these NK-cell receptors, indicating different cell origin from NK and innate-like T cells. Furthermore, NKG2D expression showed a negative impact on survival among the 22 examined cases, which mainly received the standard chemotherapy regimen (log-rank test, P = .024). We propose that the presence of activating NK-cell receptors may provide new insights into understanding peripheral T-cell lymphomas and characterizing them as innate-like T-cell neoplasm. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Altered Natural Killer Cell Subsets in Seropositive Arthralgia and Early Rheumatoid Arthritis Are Associated with Autoantibody Status.

PubMed

Chalan, Paulina; Bijzet, Johan; Kroesen, Bart-Jan; Boots, Annemieke M H; Brouwer, Elisabeth

2016-06-01

The role of natural killer (NK) cells in the immunopathogenesis of rheumatoid arthritis (RA) is unclear. Therefore, numerical and functional alterations of CD56(dim) and CD56(bright) NK cells in the early stages of RA development were studied. Whole blood samples from newly diagnosed, treatment-naive, seropositive (SP) and seronegative (SN) patients with RA (SP RA, n = 45 and SN RA, n = 12), patients with SP arthralgia (n = 30), and healthy controls (HC, n = 41) were assessed for numbers and frequencies of T cells, B cells, and NK cells. SP status was defined as positive for anticyclic citrullinated peptide antibodies (anti-CCP) and/or rheumatoid factor (RF). Peripheral blood mononuclear cells were used for further analysis of NK cell phenotype and function. Total NK cell numbers were decreased in SP RA and SP arthralgia but not in SN RA. Also, NK cells from SP RA showed a decreased potency for interferon-γ (IFN-γ) production. A selective decrease of CD56(dim), but not CD56(bright), NK cells in SP RA and SP arthralgia was observed. This prompted investigation of CD16 (FcγRIIIa) triggering in NK cell apoptosis and cytokine expression. In vitro, CD16 triggering induced apoptosis of CD56(dim) but not CD56(bright) NK cells from HC. This apoptosis was augmented by adding interleukin 2 (IL-2). Also, CD16 triggering in the presence of IL-2 stimulated IFN-γ and tumor necrosis factor-α expression by CD56(dim) NK cells. The decline of CD56(dim) NK cells in SP arthralgia and SP RA and the in vitro apoptosis of CD56(dim) NK cells upon CD16 triggering suggest a functional role of immunoglobulin G-containing autoantibody (anti-CCP and/or RF)-immune complexes in this process. Moreover, CD16-triggered cytokine production by CD56(dim) NK cells may contribute to systemic inflammation as seen in SP arthralgia and SP RA.

Intrinsic hyporesponsiveness of invariant natural killer T cells precedes the onset of lupus

PubMed Central

Yang, J-Q; Kim, P J; Halder, R C; Singh, R R

2013-01-01

Patients with systemic lupus erythematosus (SLE) display reduced numbers and functions of invariant natural killer T (iNK T) cells, which are restored upon treatment with corticosteroids and rituximab. It is unclear whether the iNK T cell insufficiency is a consequence of disease or is a primary abnormality that precedes the onset of disease. To address this, we analysed iNK T cell function at different stages of disease development using the genetically lupus-susceptible NZB × NZW F1 (BWF1) model. We found that iNK T cell in-vivo cytokine responses to an iNK T cell ligand α-galactosylceramide (α-GalCer) were lower in BWF1 mice than in non-autoimmune BALB/c and major histocompatibility complex (MHC)-matched NZB × N/B10.PL F1 mice, although iNK T cell numbers in the periphery were unchanged in BWF1 mice compared to control mice. Such iNK T cell hyporesponsiveness in BWF1 mice was detected at a young age long before the animals exhibited any sign of autoimmunity. In-vivo activation of iNK T cells is known to transactivate other immune cells. Such transactivated T and B cell activation markers and/or cytokine responses were also lower in BWF1 mice than in BALB/c controls. Finally, we show that iNK T cell responses were markedly deficient in the NZB parent but not in NZW parent of BWF1 mice, suggesting that BWF1 might inherit the iNK T cell defect from NZB mice. Thus, iNK T cells are functionally insufficient in lupus-prone BWF1 mice. Such iNK T cell insufficiency precedes the onset of disease and may play a pathogenic role during early stages of disease development in SLE. PMID:23607366

Natural Killer Cells and Helicobacter pylori Infection: Bacterial Antigens and Interleukin-12 Act Synergistically To Induce Gamma Interferon Production

PubMed Central

Yun, Cheol H.; Lundgren, Anna; Azem, Josef; Sjöling, Åsa; Holmgren, Jan; Svennerholm, Ann-Mari; Lundin, B. Samuel

2005-01-01

Helicobacter pylori is known to induce a local immune response, which is characterized by activation of lymphocytes and the production of IFN-γ in the stomach mucosa. Since not only T cells, but also natural killer (NK) cells, are potent producers of gamma interferon (IFN-γ), we investigated whether NK cells play a role in the immune response to H. pylori infection. Our results showed that NK cells were present in both the gastric and duodenal mucosae but that H. pylori infection did not affect the infiltration of NK cells into the gastrointestinal area. Furthermore, we could show that NK cells could be activated directly by H. pylori antigens, as H. pylori bacteria, as well as lysate from H. pylori, induced the secretion of IFN-γ by NK cells. NK cells were also activated without direct contact when separated from the bacteria by an epithelial cell layer, indicating that the activation of NK cells by H. pylori can also occur in vivo, in the infected stomach mucosa. Moreover, the production of IFN-γ by NK cells was greatly enhanced when a small amount of interleukin-12 (IL-12) was added, and this synergistic effect was associated with increased expression of the IL-12 receptor β2. It was further evident that bacterial lysate alone was sufficient to induce the activation of cytotoxicity-related molecules. In conclusion, we demonstrated that NK cells are present in the gastroduodenal mucosa of humans and that NK cells produce high levels of IFN-γ when stimulated with a combination of H. pylori antigen and IL-12. We propose that NK cells play an active role in the local immune response to H. pylori infection. PMID:15731046

NK cell heparanase controls tumor invasion and immune surveillance

PubMed Central

Putz, Eva M.; Mayfosh, Alyce J.; Barkauskas, Deborah S.; Nakamura, Kyohei; Town, Liam; Goodall, Katharine J.; Yee, Dean Y.; Poon, Ivan K.H.; Baschuk, Nikola; Souza-Fonseca-Guimaraes, Fernando; Hulett, Mark D.; Smyth, Mark J.

2017-01-01

NK cells are highly efficient at preventing cancer metastasis but are infrequently found in the core of primary tumors. Here, have we demonstrated that freshly isolated mouse and human NK cells express low levels of the endo-β-D-glucuronidase heparanase that increase upon NK cell activation. Heparanase deficiency did not affect development, differentiation, or tissue localization of NK cells under steady-state conditions. However, mice lacking heparanase specifically in NK cells (Hpsefl/fl NKp46-iCre mice) were highly tumor prone when challenged with the carcinogen methylcholanthrene (MCA). Hpsefl/fl NKp46-iCre mice were also more susceptible to tumor growth than were their littermate controls when challenged with the established mouse lymphoma cell line RMA-S-RAE-1β, which overexpresses the NK cell group 2D (NKG2D) ligand RAE-1β, or when inoculated with metastatic melanoma, prostate carcinoma, or mammary carcinoma cell lines. NK cell invasion of primary tumors and recruitment to the site of metastasis were strictly dependent on the presence of heparanase. Cytokine and immune checkpoint blockade immunotherapy for metastases was compromised when NK cells lacked heparanase. Our data suggest that heparanase plays a critical role in NK cell invasion into tumors and thereby tumor progression and metastases. This should be considered when systemically treating cancer patients with heparanase inhibitors, since the potential adverse effect on NK cell infiltration might limit the antitumor activity of the inhibitors. PMID:28581441

Preparation of Cytokine-activated NK Cells for Use in Adoptive Cell Therapy in Cancer Patients: Protocol Optimization and Therapeutic Potential.

PubMed

van Ostaijen-ten Dam, Monique M; Prins, Henk-Jan; Boerman, Gerharda H; Vervat, Carly; Pende, Daniela; Putter, Hein; Lankester, Arjan; van Tol, Maarten J D; Zwaginga, Jaap J; Schilham, Marco W

2016-01-01

Cell-based immunotherapy using donor-derived natural killer (NK) cells after allogeneic hematopoietic stem cell transplantation may be an attractive treatment of residual leukemia. This study aimed to optimize clinical grade production of a cytokine-activated NK-cell product. NK cells were isolated either by double depletion (CD3(-), CD19(-)) or by sequential depletion and enrichment (CD3(-,) CD56(+)) via CliniMACS from leukapheresis material and cultured in vitro with interleukin (IL)-2 or IL-15. Both NK cell isolation procedures yielded comparable recovery of NK cells and levels of T-cell contamination. After culture with cytokines, the CD3(-)CD56(+) procedure resulted in NK cells of higher purity, that is, less T cells and monocytes, higher viability, and a slightly higher yield than the CD3(-)CD19- procedure. CD69, NKp44, and NKG2A expression were higher on CD3(-)CD56(+) products, whereas lysis of Daudi cells was comparable. Five days of culture led to higher expression of CD69, NKp44, and NKp30 and lysis of K562 and Daudi cell lines. Although CD69 expression and lysis of Daudi cells were slightly higher in cultures with IL-2, T-cell contamination was lower with IL-15. Therefore, further experiments were performed with CD3(-)CD56(+) products cultured with IL-15. Cryopreservation of IL-15-activated NK cells resulted in a loss of cytotoxicity (>92%), whereas thawing of isolated, uncultured NK cells followed by culture with IL-15 yielded cells with about 43% of the original lytic activity. Five-day IL-15-activated NK cells lysed tumor target cell lines and primary leukemic blasts, providing the basis for NK cell–based immunotherapeutic strategies in a clinical setting.

Negative feedback on the effects of stem cell factor on hematopoiesis is partly mediated through neutral endopeptidase activity on substance P: a combined functional and proteomic study.

PubMed

Joshi, D D; Dang, A; Yadav, P; Qian, J; Bandari, P S; Chen, K; Donnelly, R; Castro, T; Gascon, P; Haider, A; Rameshwar, P

2001-11-01

Hematopoietic regulation is a complex but dynamic process regulated by intercellular and intracellular interactions within the bone marrow (BM) microenvironment. Through neurokinin-1 (NK-1) and NK-2 receptors, peptides (eg, substance P [SP]) encoded by the preprotachykinin-I gene mediate distinct hematopoietic effects. Cytokines, associated with hematopoietic stimulation, and SP regulate the expression of each other in BM mesenchymal and immune cells. Neutral endopeptidase (NEP) uses SP as a substrate to produce SP(1-4), which inhibits the proliferation of matured myeloid progenitor. This study determines whether the degradation of SP to SP(1-4) by endogenous NEP in BM stroma could be a feedback on hematopoietic stimulation by stem cell factor (SCF). SP(1-4) induced the production of transforming growth factor (TGF)-beta and tumor necrosis factor-alpha in BM stroma. TGF-beta production accounted for part of the inhibitory effects by SP(1-4) on the proliferation of early (granulocyte-macrophage colony-forming units) and late (long-term culture-initiating cells) hematopoietic progenitors. Enzyme-linked immunosorbent assays and/or protein-chip arrays indicated a timeline change of SP to SP(1-4) in BM stroma stimulated with SCF, which correlated with increase in NEP messenger RNA. Since SP and its fragment, SP(1-4), interact with the same receptor to mediate opposing hematopoietic effects, 2 interactive studies were done to understand the dual responses of NK-1: (1) a 3-dimensional molecular model of NK-1 and SP and (2) screening of a random dodecapeptide library for SP(1-4) interacting sites. The effects of SP(1-4) on hematopoietic progenitors and the timeline change of SP to SP(1-4), together with the 3-dimensional model, provide a partial explanation for the feedback on the stimulatory effects of SCF and SP on hematopoiesis.

NK cells and poxvirus infection

PubMed Central

Burshtyn, Deborah N.

2013-01-01

In recent years, our understanding of the role of natural killer (NK) cells in the response to viral infection has grown rapidly. Not only do we realize viruses have many immune-evasion strategies to escape NK cell responses, but that stimulation of NK cell subsets during an antiviral response occurs through receptors seemingly geared directly at viral products and that NK cells can provide a memory response to viral pathogens. Tremendous knowledge has been gained in this area through the study of herpes viruses, but appreciation for the significance of NK cells in the response to other types of viral infections is growing. The function of NK cells in defense against poxviruses has emerged over several decades beginning with the early seminal studies showing the role of NK cells and the NK gene complex in susceptibility of mouse strains to ectromelia, a poxvirus pathogen of mice. More recently, greater understanding has emerged of the molecular details of the response. Given that human diseases caused by poxviruses can be as lethal as smallpox or as benign as Molluscum contagiosum, and that vaccinia virus, the prototypic member of the pox family, persists as a mainstay of vaccine design and has potential as an oncolytic virus for tumor therapy, further research in this area remains important. This review focuses on recent advances in understanding the role of NK cells in the immune response to poxviruses, the receptors involved in activation of NK cells during poxvirus infection, and the viral evasion strategies poxviruses employ to avoid the NK response. PMID:23372568

Activation of NK Cells in Mixed Cultures of Wharton's Jelly Mesenchymal Stromal Cells and Peripheral Blood Lymphocytes.

PubMed

Svirshchevskaya, E V; Poltavtsev, A M; Os'mak, G Zh; Poltavtseva, R A

2018-01-01

Mesenchymal stromal cells possess immunosuppressive properties that might be used for the therapy of inflammatory diseases of various geneses. The effects of mesenchymal stromal cells depend on their lifetime in the recipient tissues. During heterologous transplantation, mesenchymal stromal cells are eliminated by NK cells. We studied NK cell formation in mixed cultures of Wharton's jelly mesenchymal stromal cells and peripheral blood lymphocytes from an autologous donor. Lymphocytes were activated by a mitogen or IL-2. The lifetime of mesenchymal stromal cells was estimated by MTT test. Cytotoxic activity and phenotype of NK cells were evaluated by flow cytometry. It was found that activation of NK cells depended on IL-2 and was registered on day 2 of incubation with IL-2. In cultures with mitogen-activated lymphocytes, cytotoxicity was observed after 5-6 days. Cytotoxicity of NK correlated with significant decrease in CD16+ and increase in CD56+ NK and with reduction of mesenchymal stromal cell viability. Thus, the main mechanism of elimination of mesenchymal stromal cells is cytotoxicity of NK cells that depended on IL-2 production.

Role of adaptive and innate immune cells in chronic fatigue syndrome/myalgic encephalomyelitis.

PubMed

Brenu, Ekua Weba; Huth, Teilah K; Hardcastle, Sharni L; Fuller, Kirsty; Kaur, Manprit; Johnston, Samantha; Ramos, Sandra B; Staines, Don R; Marshall-Gradisnik, Sonya M

2014-04-01

Perturbations in immune processes are a hallmark of a number of autoimmune and inflammatory disorders. Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is an inflammatory disorder with possible autoimmune correlates, characterized by reduced NK cell activity, elevations in regulatory T cells (Tregs) and dysregulation in cytokine levels. The purpose of this article is to examine innate and adaptive immune cell phenotypes and functional characteristics that have not been previously examined in CFS/ME patients. Thirty patients with CFS/ME and 25 non-fatigued controls were recruited for this study. Whole blood samples were collected from all participants for the assessment of cell phenotypes, functional properties, receptors, adhesion molecules, antigens and intracellular proteins using flow cytometric protocols. The cells investigated included NK cells, dendritic cells, neutrophils, B cells, T cells, γδT cells and Tregs. Significant changes were observed in B-cell subsets, Tregs, CD4(+)CD73(+)CD39(+) T cells, cytotoxic activity, granzyme B, neutrophil antigens, TNF-α and IFN-γ in the CFS/ME patients in comparison with the non-fatigued controls. Alterations in B cells, Tregs, NK cells and neutrophils suggest significant impairments in immune regulation in CFS/ME and these may have similarities to a number of autoimmune disorders.

Selective Inhibition of Tumor Growth by Clonal NK Cells Expressing an ErbB2/HER2-Specific Chimeric Antigen Receptor

PubMed Central

Schönfeld, Kurt; Sahm, Christiane; Zhang, Congcong; Naundorf, Sonja; Brendel, Christian; Odendahl, Marcus; Nowakowska, Paulina; Bönig, Halvard; Köhl, Ulrike; Kloess, Stephan; Köhler, Sylvia; Holtgreve-Grez, Heidi; Jauch, Anna; Schmidt, Manfred; Schubert, Ralf; Kühlcke, Klaus; Seifried, Erhard; Klingemann, Hans G; Rieger, Michael A; Tonn, Torsten; Grez, Manuel; Wels, Winfried S

2015-01-01

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy. PMID:25373520

STATs in NK-Cells: The Good, the Bad, and the Ugly.

PubMed

Gotthardt, Dagmar; Sexl, Veronika

2016-01-01

Natural killer (NK)-cells are major players in the fight against viral infections and transformed cells, but there is increasing evidence attributing a disease-promoting role to NK-cells. Cytokines present in the tumor microenvironment shape NK-cell maturation, function, and effector responses. Many cytokines signal via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway that is also frequently altered and constitutively active in a broad range of tumor cells. As a consequence, there are currently major efforts to develop therapeutic strategies to target this pathway. Therefore, it is of utmost importance to understand the role and contributions of JAK-STAT molecules in NK-cell biology-only this knowledge will allow us to predict effects of JAK-STAT inhibition for NK-cell functions and to successfully apply precision medicine. We will review the current knowledge on the role of JAK-STAT signaling for NK-cell functions and discuss conditions involved in the switch from NK-cell tumor surveillance to disease promotion.

Gene expression analysis of hypersensitivity to mosquito bite, chronic active EBV infection and NK/T-lymphoma/leukemia.

PubMed

Washio, Kana; Oka, Takashi; Abdalkader, Lamia; Muraoka, Michiko; Shimada, Akira; Oda, Megumi; Sato, Hiaki; Takata, Katsuyoshi; Kagami, Yoshitoyo; Shimizu, Norio; Kato, Seiichi; Kimura, Hiroshi; Nishizaki, Kazunori; Yoshino, Tadashi; Tsukahara, Hirokazu

2017-11-01

The human herpes virus, Epstein-Barr virus (EBV), is a known oncogenic virus and plays important roles in life-threatening T/NK-cell lymphoproliferative disorders (T/NK-cell LPD) such as hypersensitivity to mosquito bite (HMB), chronic active EBV infection (CAEBV), and NK/T-cell lymphoma/leukemia. During the clinical courses of HMB and CAEBV, patients frequently develop malignant lymphomas and the diseases passively progress sequentially. In the present study, gene expression of CD16 (-) CD56 (+) -, EBV (+) HMB, CAEBV, NK-lymphoma, and NK-leukemia cell lines, which were established from patients, was analyzed using oligonucleotide microarrays and compared to that of CD56 bright CD16 dim/- NK cells from healthy donors. Principal components analysis showed that CAEBV and NK-lymphoma cells were relatively closely located, indicating that they had similar expression profiles. Unsupervised hierarchal clustering analyses of microarray data and gene ontology analysis revealed specific gene clusters and identified several candidate genes responsible for disease that can be used to discriminate each category of NK-LPD and NK-cell lymphoma/leukemia.

Engineering NK Cells Modified With an EGFRvIII-specific Chimeric Antigen Receptor to Overexpress CXCR4 Improves Immunotherapy of CXCL12/SDF-1α-secreting Glioblastoma.

PubMed

Müller, Nadja; Michen, Susanne; Tietze, Stefanie; Töpfer, Katrin; Schulte, Alexander; Lamszus, Katrin; Schmitz, Marc; Schackert, Gabriele; Pastan, Ira; Temme, Achim

2015-06-01

Natural killer (NK) cells are promising effector cells for adjuvant immunotherapy of cancer. So far, several preclinical studies have shown the feasibility of gene-engineered NK cells, which upon expression of chimeric antigen receptors (CARs) are redirected to otherwise NK cell-resistant tumors. Yet, we reasoned that the efficiency of an immunotherapy using CAR-modified NK cells critically relies on efficient migration to the tumor site and might be improved by the engraftment of a receptor specific for a chemokine released by the tumor. On the basis of the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an epidermal growth factor variant III (EGFRvIII)-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII glioblastoma cells in vitro and to established subcutaneous U87-MG tumor xenografts. So far, infusion of NK cells with expression of MR1.1-DAP12 caused a moderate but significantly delayed tumor growth and increased median survival time when compared with NK cells transduced with an ITAM-defective CAR. Notably, the further genetic engineering of these EGFRvIII-specific NK cells with the chemokine receptor CXCR4 conferred a specific chemotaxis to CXCL12/SDF-1α secreting U87-MG glioblastoma cells. Moreover, the administration of such NK cells resulted in complete tumor remission in a number of mice and a significantly increased survival when compared with the treatment of xenografts with NK cells expressing only the EGFRvIII-specific CAR or mock control. We conclude that chemokine receptor-engineered NK cells with concomitant expression of a tumor-specific CAR are a promising tool to improve adoptive tumor immunotherapy.

Ex Vivo Expanded Natural Killer Cells Demonstrate Robust Proliferation In Vivo In High-Risk Relapsed Multiple Myeloma Patients

PubMed Central

Szmania, Susann; Lapteva, Natalia; Garg, Tarun; Greenway, Amy; Lingo, Joshuah; Nair, Bijay; Stone, Katie; Woods, Emily; Khan, Junaid; Stivers, Justin; Panozzo, Susan; Campana, Dario; Bellamy, William T.; Robbins, Molly; Epstein, Joshua; Yaccoby, Shmuel; Waheed, Sarah; Gee, Adrian; Cottler-Fox, Michele; Rooney, Cliona; Barlogie, Bart; van Rhee, Frits

2015-01-01

Highly activated/expanded natural killer (NK) cells can be generated via stimulation with the HLA-deficient cell line K562 genetically modified to express 41BB-ligand and membrane-bound interleukin (IL)15. We tested the safety, persistence and activity of expanded NK cells generated from myeloma patients (auto-NK) or haplo-identical family donors (allo-NK) in heavily pretreated patients with high-risk relapsing myeloma. The preparative regimen comprised bortezomib only or bortezomib and immunosuppression with cyclophosphamide, dexamethasone and fludarabine. NK cells were shipped overnight either cryopreserved or fresh. In 8 patients, up to 1×108 NK cells/kg were infused on day 0 and followed by daily administrations of IL2. Significant in vivo expansion was observed only in the 5 patients receiving fresh products, peaking at or near day 7, with the highest NK cell counts in 2 subjects who received cells produced in a high concentration of IL2 (500 units/mL). Seven days after infusion, donor NK cells comprised > 90% of circulating leukocytes in fresh allo-NK cell recipients, and cytolytic activity against allogeneic myeloma targets was retained in vitro. Among the 7 evaluable patients, there were no serious adverse events that could be related to NK cell infusion. One patient had a partial response and in another the tempo of disease progression decreased; neither patient required further therapy for 6 months. In the 5 remaining patients, disease progression was not affected by NK cell infusion. In conclusion, infusion of large numbers of expanded NK cells was feasible and safe; infusing fresh cells was critical to their expansion in vivo. PMID:25415285

NK-92 cell, another ideal carrier for chimeric antigen receptor.

PubMed

Wang, Wan-Ning; Zhou, Guang-Yu; Zhang, Wen-Long

2017-08-01

The remarkable clinical outcomes of the treatment for B-cell malignancies through the application of CD19 chimeric antigen receptor T (CAR-T) cells have made adoptive immunotherapy with genetically modified immune effector cells a hotspot in the field of antitumor. However, numerous toxicities of CAR-T cells have been identified. Thus, some studies have resorted to another cytotoxic cell, NK-92 cell, to reach for better efficacy with minimal toxicity. Preclinical studies have confirmed the safety and feasibility of the genetically modified NK-92 cells with highly specific cytotoxicity in vitro and in vivo. Therefore, it is expected that NK-92 cell becomes another ideal carrier for CAR for its unique advantages over primary NK cells, parental NK-92 cells and autologous T cells.

Growth and apoptosis of human natural killer cell neoplasms: role of interleukin-2/15 signaling.

PubMed

Yamasaki, Satoshi; Maeda, Motoi; Ohshima, Koichi; Kikuchi, Masahiro; Otsuka, Teruhisa; Harada, Mine

2004-10-01

Interleukin (IL)-15 plays an important role in the survival of human natural killer (NK) cells. We investigated IL-2/15 signaling in NK cell neoplasms from five patients and in five cell lines (NK-92, KHYG-1, SNK-6, HANK1 and MOTN-1) compared to mature peripheral NK cells from 10 healthy subjects. Apoptosis of NK cell lines was prevented by addition of IL-15 in vitro. Blocking IL-2/15Rbeta on IL-2-stimulated NK-92 cells resulted in reduced expression of Bcl-X(L) and phosphorylated Stat5, which paralleled early apoptosis without altering Bcl-2 expression. These data add IL-2/15Rbeta to the list of factors important for the survival of NK cell neoplasms.

HLA-E upregulation on IFN-gamma-activated AML blasts impairs CD94/NKG2A-dependent NK cytolysis after haplo-mismatched hematopoietic SCT.

PubMed

Nguyen, S; Beziat, V; Dhedin, N; Kuentz, M; Vernant, J P; Debre, P; Vieillard, V

2009-05-01

Natural killer (NK) cells generated after haploidentical hematopoietic SCT in patients with AML are characterized by specific phenotypic features and impaired functioning that may affect transplantation outcome. We show that IFN-gamma produced by immature CD56(bright) NK cells upregulates cell surface expression of HLA-E on AML blasts and that this upregulation protects leukemic cells from NK-mediated cell lysis through the mediation of CD94/NKG2A, an inhibitory receptor overexpressed on NK cells after haploidentical SCT. Two years after transplantation, however, maturing NK cells were functionally active, as evidenced by high cytotoxicity and poor IFN-gamma production. This implies that maturation of NK cells is the key to improved immune responses and transplantation outcome.

The role of substance P in inflammatory disease.

PubMed

O'Connor, Terence M; O'Connell, Joseph; O'Brien, Darren I; Goode, Triona; Bredin, Charles P; Shanahan, Fergus

2004-11-01

The diffuse neuroendocrine system consists of specialised endocrine cells and peptidergic nerves and is present in all organs of the body. Substance P (SP) is secreted by nerves and inflammatory cells such as macrophages, eosinophils, lymphocytes, and dendritic cells and acts by binding to the neurokinin-1 receptor (NK-1R). SP has proinflammatory effects in immune and epithelial cells and participates in inflammatory diseases of the respiratory, gastrointestinal, and musculoskeletal systems. Many substances induce neuropeptide release from sensory nerves in the lung, including allergen, histamine, prostaglandins, and leukotrienes. Patients with asthma are hyperresponsive to SP and NK-1R expression is increased in their bronchi. Neurogenic inflammation also participates in virus-associated respiratory infection, non-productive cough, allergic rhinitis, and sarcoidosis. SP regulates smooth muscle contractility, epithelial ion transport, vascular permeability, and immune function in the gastrointestinal tract. Elevated levels of SP and upregulated NK-1R expression have been reported in the rectum and colon of patients with inflammatory bowel disease (IBD), and correlate with disease activity. Increased levels of SP are found in the synovial fluid and serum of patients with rheumatoid arthritis (RA) and NK-1R mRNA is upregulated in RA synoviocytes. Glucocorticoids may attenuate neurogenic inflammation by decreasing NK-1R expression in epithelial and inflammatory cells and increasing production of neutral endopeptidase (NEP), an enzyme that degrades SP. Preventing the proinflammatory effects of SP using tachykinin receptor antagonists may have therapeutic potential in inflammatory diseases such as asthma, sarcoidosis, chronic bronchitis, IBD, and RA. In this paper, we review the role that SP plays in inflammatory disease. Copyright 2004 Wiley-Liss, Inc.

Natural killer cells inhibit oxaliplatin-resistant colorectal cancer by repressing WBSCR22 via upregulating microRNA-146b-5p.

PubMed

Zhao, Haiyan; Su, Wuyun; Kang, Qingmei; Xing, Ze; Lin, Xue; Wu, Zhongjun

2018-01-01

Natural killer (NK) cells have exhibited promising efficacy in inhibiting cancer growth. We aimed to explorer the effect of NK cells on oxaliplatin-resistant colorectal cancer and the underlying molecular mechanism. Oxaliplatin-resistant colorectal cancer cell lines were co-cultured with NK cells to evaluate the effect on viability, proliferation, migration and invasion in vitro . Oxaliplatin-resistant colorectal cancer cells were also co-injected with NK cells into mice to establish xenograft tumor model, to assess the in vivo effect of NK cells on tumorigenesis of the oxaliplatin-resistant colorectal cancer cells. Expression of WBSCR22 gene was assessed in the oxaliplatin-resistant colorectal cancer cells following NK cell treatment to elucidate the mechanism. NK cell treatment significantly reduces growth of oxaliplatin-resistant colorectal cancer cells both in vitro and in vivo , as well as reduced WBSCR22 expression. MicroRNAs potentially targeting WBSCR22 were analyzed, and microRNA-146b-5p was found to be significantly upregulated following NK cell treatment. MicroRNA-146b-5p directly targeted WBSCR22 mRNA 3'-UTR to inhibit its expression, which was required for NK cell-induced inhibition of oxaliplatin-resistant colorectal cancer cell lines. NK cells inhibit oxaliplatin-resistant colorectal cancer by repressing WBSCR22 via upregulating microRNA-146b-5p, both of which could serve as candidates for targeted therapy against oxaliplatin-resistant colorectal cancer.

IL-2 induces a WAVE2-dependent pathway for actin reorganization that enables WASp-independent human NK cell function.

PubMed

Orange, Jordan S; Roy-Ghanta, Sumita; Mace, Emily M; Maru, Saumya; Rak, Gregory D; Sanborn, Keri B; Fasth, Anders; Saltzman, Rushani; Paisley, Allison; Monaco-Shawver, Linda; Banerjee, Pinaki P; Pandey, Rahul

2011-04-01

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency associated with an increased susceptibility to herpesvirus infection and hematologic malignancy as well as a deficiency of NK cell function. It is caused by defective WAS protein (WASp). WASp facilitates filamentous actin (F-actin) branching and is required for F-actin accumulation at the NK cell immunological synapse and NK cell cytotoxicity ex vivo. Importantly, the function of WASp-deficient NK cells can be restored in vitro after exposure to IL-2, but the mechanisms underlying this remain unknown. Using a WASp inhibitor as well as cells from patients with WAS, we have defined a direct effect of IL-2 signaling upon F-actin that is independent of WASp function. We found that IL-2 treatment of a patient with WAS enhanced the cytotoxicity of their NK cells and the F-actin content at the immunological synapses formed by their NK cells. IL-2 stimulation of NK cells in vitro activated the WASp homolog WAVE2, which was required for inducing WASp-independent NK cell function, but not for baseline activity. Thus, WAVE2 and WASp define parallel pathways to F-actin reorganization and function in human NK cells; although WAVE2 was not required for NK cell innate function, it was accessible through adaptive immunity via IL-2. These results demonstrate how overlapping cytoskeletal activities can utilize immunologically distinct pathways to achieve synonymous immune function.

IL-2 induces a WAVE2-dependent pathway for actin reorganization that enables WASp-independent human NK cell function

PubMed Central

Orange, Jordan S.; Roy-Ghanta, Sumita; Mace, Emily M.; Maru, Saumya; Rak, Gregory D.; Sanborn, Keri B.; Fasth, Anders; Saltzman, Rushani; Paisley, Allison; Monaco-Shawver, Linda; Banerjee, Pinaki P.; Pandey, Rahul

2011-01-01

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency associated with an increased susceptibility to herpesvirus infection and hematologic malignancy as well as a deficiency of NK cell function. It is caused by defective WAS protein (WASp). WASp facilitates filamentous actin (F-actin) branching and is required for F-actin accumulation at the NK cell immunological synapse and NK cell cytotoxicity ex vivo. Importantly, the function of WASp-deficient NK cells can be restored in vitro after exposure to IL-2, but the mechanisms underlying this remain unknown. Using a WASp inhibitor as well as cells from patients with WAS, we have defined a direct effect of IL-2 signaling upon F-actin that is independent of WASp function. We found that IL-2 treatment of a patient with WAS enhanced the cytotoxicity of their NK cells and the F-actin content at the immunological synapses formed by their NK cells. IL-2 stimulation of NK cells in vitro activated the WASp homolog WAVE2, which was required for inducing WASp-independent NK cell function, but not for baseline activity. Thus, WAVE2 and WASp define parallel pathways to F-actin reorganization and function in human NK cells; although WAVE2 was not required for NK cell innate function, it was accessible through adaptive immunity via IL-2. These results demonstrate how overlapping cytoskeletal activities can utilize immunologically distinct pathways to achieve synonymous immune function. PMID:21383498

Interferon-gamma: biologic functions and HCV therapy (type I/II) (1 of 2 parts).

PubMed

Gattoni, A; Parlato, A; Vangieri, B; Bresciani, M; Derna, R

2006-01-01

This review is aimed at exhaustively presenting and discussing the interferon-gamma (IFN-gamma), a cytokine that plays an important role in inducing and modulating an array of immune responses. A review of the most significant and recent clinical trials was performed. Although IFN-gamma has some antiviral activity, it is much less active in this regard than type I IFNs. IFN-gamma is involved in the regulation of nearly all phases of the immune and inflammatory responses, including the activation and differentiation of T cells, B cells, NK cells, macrophages, and others. It is therefore best regarded as a distint immunoregulatory cytokine. IFN-gamma secretion is a hallmark of Th1 lymphocytes. It is also secreted by nearly all CD8 T cells, by some Th0 cells, and by NK cells. Each of these cell types secretes IFN-gamma only when activated, usually as part of immune response and especially in response to IL-2 and IL-12. IFN-gamma production is inhibited by IL-4, IL-10, TGFbeta, glucocorticoids, cyclosporin A and FK506. Nearly all cell types express the heterodimeric receptor for IFN-beta and respond to this cytokine by increasing the surface expression of class I MHC proteins. As a result, virtually any cell in the vicinity of an IFN-beta-secreting cell becomes more efficient at presenting endogenous antigens and hence a better target for cytotoxic killing if it harbors an intracellular pathogen. Unlike the type I IFNs, IFN-gamma also increases the expression of class II MHC proteins on professional APCs, and so promotes antigen presentation to helper T cells as well. It also induces de novo expression of class II MHC proteins on venular endothelial cells and on some other epithelial and connective tissue cells that do not otherwise express them, thus enabling these cell types to function as temporary APCs at sites of intense immune reactions. The effector functions of NK cells are to lyse virus-infected cells and to secrete IFN-gamma, which activates macrofages to destroy phagocytosed microbes. The mechanism of NK cell-mediates cytolysis is essentially the same as that of cytolysis by CTLS. NK cells lyse virally infected cells before antigen specific CTLS came become fully active, that is, during the first few days after viral infection. NK cells are expanded and activated by cytokines of innate immunity, such as IL-12 and IL-15, and they kill infected cells, especially those that display reduced levels of class I molecoles. Some tumors, especially those of hematopoietic origin, are targets of NK cells, perlevels or types of class I MHC molecules. Therefore, IFN-gamma serves critical functions in innate immunity and in specific cell-mediated immunity (in addition, IFN activates neutrophilis and stimulates the cytolitic activity of NK cells). Many IFNs-gamma induced effects result in heigtened immune surveillance. IFN-gamma is a remarkable cytokine that orchestrates many distinct cellular programs through transcriptional control over large numbers of genes. Many IFNs-gamma-induced effects resulting in heightend immune surveillance and immune system function during infection have been discussed in this review. As the pathogens (microorganism with the potential to cause tissue injury or disease) augment local IFN-gamma production, and IFN-gamma augments the immune system response, an important function of IFN-gamma during in vivo infection is suggested. IFN-gamma is primarily secreted by activated T cells and natural killer cells, and can promote macrophage activation, mediate antiviral e antibacterial immunity, enhance antigen presentation, orchestrate activation of the innate immune system, coordinate lymphocyte-endothelium interaction, regulate Th1/Th2 balance, and control cellular proliferation and apoptosis.

TUSC2 Immunogene Therapy Synergizes with Anti-PD-1 through Enhanced Proliferation and Infiltration of Natural Killer Cells in Syngeneic Kras-Mutant Mouse Lung Cancer Models.

PubMed

Meraz, Ismail M; Majidi, Mourad; Cao, Xiaobo; Lin, Heather; Li, Lerong; Wang, Jing; Baladandayuthapani, Veera; Rice, David; Sepesi, Boris; Ji, Lin; Roth, Jack A

2018-02-01

Expression of the multikinase inhibitor encoded by the tumor suppressor gene TUSC2 (also known as FUS1 ) is lost or decreased in non-small cell lung carcinoma (NSCLC). TUSC2 delivered systemically by nanovesicles has mediated tumor regression in clinical trials. Because of the role of TUSC2 in regulating immune cells, we assessed TUSC2 efficacy on antitumor immune responses alone and in combination with anti-PD-1 in two Kras -mutant syngeneic mouse lung cancer models. TUSC2 alone significantly reduced tumor growth and prolonged survival compared with anti-PD-1. When combined, this effect was significantly enhanced, and correlated with a pronounced increases in circulating and splenic natural killer (NK) cells and CD8 + T cells, and a decrease in regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and T-cell checkpoint receptors PD-1, CTLA-4, and TIM-3. TUSC2 combined with anti-PD-1 induced tumor infiltrating more than NK and CD8 + T cells and fewer MDSCs and Tregs than each agent alone, both in subcutaneous tumor and in lung metastases. NK-cell depletion abrogated the antitumor effect and Th1-mediated immune response of this combination, indicating that NK cells mediate TUSC2/anti-PD-1 synergy. Release of IL15 and IL18 cytokines and expression of the IL15Rα chain and IL18R1 were associated with NK-cell activation by TUSC2. Immune response-related gene expression in the tumor microenvironment was altered by combination treatment. These data provide a rationale for immunogene therapy combined with immune checkpoint blockade in the treatment of NSCLC. Cancer Immunol Res; 6(2); 163-77. ©2018 AACR . ©2018 American Association for Cancer Research.

Activation of mouse liver natural killer cells and NK1.1(+) T cells by bacterial superantigen-primed Kupffer cells.

PubMed

Dobashi, H; Seki, S; Habu, Y; Ohkawa, T; Takeshita, S; Hiraide, H; Sekine, I

1999-08-01

Although bacterial superantigens have been well characterized as potent stimulators of T cells, their role in natural killer (NK)-type cells remains largely unknown. In the present study, we examined the effect of bacterial superantigens on mouse liver NK cells and NK1.1 Ag(+) (NK1(+)) T cells. C57BL/6 mice were intravenously injected with staphylococcal enterotoxin B (SEB) or streptococcal pyrogenic exotoxin A (SPE-A), and mononuclear cells (MNC) of various organs were obtained from mice 4 hours after being injected with superantigen. MNC were cultured for 48 hours, and interferon gamma (IFN-gamma) levels of supernatants were measured. The antitumor cytotoxicities of the liver and spleen MNC were also evaluated 24 hours after the mice were injected with superantigen. Liver MNC produced more IFN-gamma than did splenocytes, and peripheral blood and lung MNC did not produce any detectable IFN-gamma. In addition, liver MNC acquired a potent antitumor cytotoxicity by the SEB injection, and both NK cells and NK1(+)T cells but not cluster of differentiation (CD)8(+) T cells were responsible for the cytotoxicity as demonstrated by either in vivo or in vitro cell depletion experiments, and the NK-type cells were partly responsible for the increased serum IFN-gamma. Activation of liver NK-type cells was also supported by the fact that liver NK cells proportionally increased and NK1(+) T cells augmented their CD11a expressions after SEB injection. The pretreatment of mice with anti-IFN-gamma Ab and/or with anti-interleukin-12 (IL-12) Ab diminished the SEB-induced cytotoxicity of liver MNC. Furthermore, the in vivo depletion of Kupffer cells decreased the SEB-induced cytotoxicity of liver MNC. Consistent with these results, liver MNC stimulated with superantigens in the presence of Kupffer cells in vitro produced a greater amount of IFN-gamma than did the liver MNC without Kupffer cells or splenocytes. Our results suggest that bacterial superantigen-primed Kupffer cells produce IL-12 and other monokines, while also nonspecifically activating both NK cells and NK1(+) T cells to produce IFN-gamma.

GVHD prevents NK-cell-dependent leukemia and virus-specific innate immunity.

PubMed

Bunting, Mark D; Varelias, Antiopi; Souza-Fonseca-Guimaraes, Fernando; Schuster, Iona S; Lineburg, Katie E; Kuns, Rachel D; Fleming, Peter; Locke, Kelly R; Huntington, Nicholas D; Blazar, Bruce R; Lane, Steven W; Tey, Siok-Keen; MacDonald, Kelli P A; Smyth, Mark J; Degli-Esposti, Mariapia A; Hill, Geoffrey R

2017-02-02

Allogeneic bone marrow transplantation (allo-BMT) is a curative therapy for hematological malignancies, but is associated with significant complications, principally graft-versus-host disease (GVHD) and opportunistic infections. Natural killer (NK) cells mediate important innate immunity that provides a temporal bridge until the reconstruction of adaptive immunity. Here, we show that the development of GVHD after allo-BMT prevented NK-cell reconstitution, particularly within the maturing M1 and M2 NK-cell subsets in association with exaggerated activation, apoptosis, and autophagy. Donor T cells were critical in this process by limiting the availability of interleukin 15 (IL-15), and administration of IL-15/IL-15Rα or immune suppression with rapamycin could restore NK-cell reconstitution. Importantly, the NK-cell defect induced by GVHD resulted in the failure of NK-cell-dependent in vivo cytotoxicity and graft-versus-leukemia effects. Control of cytomegalovirus infection after allo-BMT was also impaired during GVHD. Thus, during GVHD, donor T cells compete with NK cells for IL-15 thereby inducing profound defects in NK-cell reconstitution that compromise both leukemia and pathogen-specific immunity. © 2017 by The American Society of Hematology.

Increased level and interferon-γ production of circulating natural killer cells in patients with scrub typhus.

PubMed

Kang, Seung-Ji; Jin, Hye-Mi; Cho, Young-Nan; Kim, Seong Eun; Kim, Uh Jin; Park, Kyung-Hwa; Jang, Hee-Chang; Jung, Sook-In; Kee, Seung-Jung; Park, Yong-Wook

2017-07-01

Natural killer (NK) cells are essential immune cells against several pathogens. Not much is known regarding the roll of NK cells in Orientia tsutsugamushi infection. Thus, this study aims to determine the level, function, and clinical relevance of NK cells in patients with scrub typhus. This study enrolled fifty-six scrub typhus patients and 56 health controls (HCs). The patients were divided into subgroups according to their disease severity. A flow cytometry measured NK cell level and function in peripheral blood. Circulating NK cell levels and CD69 expressions were significantly increased in scrub typhus patients. Increased NK cell levels reflected disease severity. In scrub typhus patients, tests showed their NK cells produced higher amounts of interferon (IFN)-γ after stimulation with interleukin (IL)-12 and IL-18 relative to those of HCs. Meanwhile, between scrub typhus patients and HCs, the cytotoxicity and degranulation of NK cells against K562 were comparable. CD69 expressions were recovered to the normal levels in the remission phase. This study shows that circulating NK cells are activated and numerically increased, and they produced more IFN-γ in scrub typhus patients.

Increased level and interferon-γ production of circulating natural killer cells in patients with scrub typhus

PubMed Central

Cho, Young-Nan; Kim, Seong Eun; Kim, Uh Jin; Park, Kyung-Hwa; Jang, Hee-Chang; Jung, Sook-In; Kee, Seung-Jung

2017-01-01

Background Natural killer (NK) cells are essential immune cells against several pathogens. Not much is known regarding the roll of NK cells in Orientia tsutsugamushi infection. Thus, this study aims to determine the level, function, and clinical relevance of NK cells in patients with scrub typhus. Methodology/Principal findings This study enrolled fifty-six scrub typhus patients and 56 health controls (HCs). The patients were divided into subgroups according to their disease severity. A flow cytometry measured NK cell level and function in peripheral blood. Circulating NK cell levels and CD69 expressions were significantly increased in scrub typhus patients. Increased NK cell levels reflected disease severity. In scrub typhus patients, tests showed their NK cells produced higher amounts of interferon (IFN)-γ after stimulation with interleukin (IL)-12 and IL-18 relative to those of HCs. Meanwhile, between scrub typhus patients and HCs, the cytotoxicity and degranulation of NK cells against K562 were comparable. CD69 expressions were recovered to the normal levels in the remission phase. Conclusions This study shows that circulating NK cells are activated and numerically increased, and they produced more IFN-γ in scrub typhus patients. PMID:28750012

Working in "NK Mode": Natural Killer Group 2 Member D and Natural Cytotoxicity Receptors in Stress-Surveillance by γδ T Cells.

PubMed

Silva-Santos, Bruno; Strid, Jessica

2018-01-01

Natural killer cell receptors (NKRs) are germline-encoded transmembrane proteins that regulate the activation and homeostasis of NK cells as well as other lymphocytes. For γδ T cells, NKRs play critical roles in discriminating stressed (transformed or infected) cells from their healthy counterparts, as proposed in the "lymphoid stress-surveillance" theory. Whereas the main physiologic role is seemingly fulfilled by natural killer group 2 member D, constitutively expressed by γδ T cells, enhancement of their therapeutic potential may rely on natural cytotoxicity receptors (NCRs), like NKp30 or NKp44, that can be induced selectively on human Vδ1 + T cells. Here, we review the contributions of NCRs, NKG2D, and their multiple ligands, to γδ T cell biology in mouse and human.

The cytotoxic action of the CD56+ fraction of cytokine-induced killer cells against a K562 cell line is mainly restricted to the natural killer cell subset.

PubMed

Chieregato, Katia; Zanon, Cristina; Castegnaro, Silvia; Bernardi, Martina; Amati, Eliana; Sella, Sabrina; Rodeghiero, Francesco; Astori, Giuseppe

2017-01-01

Cytokine-induced killer cells are polyclonal T cells generated ex vivo and comprise two main subsets: the CD56- fraction, possessing an alloreactive potential caused by T cells (CD3+CD56-), and the CD56+ fraction, characterised by a strong antitumour capacity induced by natural killer-like T cells (NK-like T, CD3+CD56+) and natural killer cells (NK, CD3-CD56+ bright). We investigated the cytotoxic action of selected CD56+ cell subpopulations against a human chronic myeloid leukaemia (K562) cell line. After immunomagnetic selection of the CD56+ cell fraction, NK bright cells (CD3-CD56+ bright) and two subsets of NK-like T cells (CD3+CD56+), called NK-like T CD56 dim and NK-like T CD56 bright, could be identified. The cytotoxic effect against K562 cells was mainly exerted by the NK bright subpopulation and resulted to be inversely correlated with the percentage of NK-like T CD56 dim cells in the culture. The lytic action appeared to be independent of cell degranulation as suggested by the lack of change in the expression of CD107a. We conclude that the cytotoxic action of CD56+ cells against a K562 cell line is mainly due to the NK cells.

Changes in endometrial natural killer cell expression of CD94, CD158a and CD158b are associated with infertility.

PubMed

McGrath, Emma; Ryan, Elizabeth J; Lynch, Lydia; Golden-Mason, Lucy; Mooney, Eoghan; Eogan, Maeve; O'Herlihy, Colm; O'Farrelly, Cliona

2009-04-01

Cycle-dependent fluctuations in natural killer (NK) cell populations in endometrium and circulation may differ, contributing to unexplained infertility. NK cell phenotypes were determined by flow cytometry in endometrial biopsies and matched blood samples. While circulating and endometrial T cell populations remained constant throughout the menstrual cycle in fertile and infertile women, circulating NK cells in infertile women increased during the secretory phase. However, increased expression of CD94, CD158b (secretory phase), and CD158a (proliferative phase) by endometrial NK cells from infertile women was observed. These changes were not reflected in the circulation. In infertile women, changes in circulating NK cell percentages are found exclusively during the secretory phase and not in endometrium; cycle-related changes in NK receptor expression are observed only in infertile endometrium. While having exciting implications for understanding NK cell function in fertility, our data emphasize the difficulty in attaching diagnostic or prognostic significance to NK cell analyses in individual patients.

[Immunoregulation by iNKT cells].

PubMed

Miyake, Sachiko

2009-06-01

NKT cells are defined as cells co-expressing of the natural killer receptors such as NK1.1 or NKR-P1A (CD161) and a T cell receptor (TCR). Although NK1.1(+) TCR(+) lymphocytes are heterogeneous, we focus on two distinct T cell subsets express invariant T cell receptor alpha chains, Valpha14-Jalpha18(Valpha14i) and Valpha19-Jalpha33(Valpha19i). Valpha14i NKT cells (Valpha24i NKT cells for human) are restricted by CD1d and Valpha19i NKT cells (Valpha7.2i NKT cells for human) are restricted by MR1 molecule. These cells emerge as an unique lymphocytes subset to bridge innate and acquired immunity. Here in this review, we discuss on the role of these cells in the regulation of autoimmunity and on the potential of therapeutic target for autoimmune diseases.

Modeling Human Natural Killer Cell Development in the Era of Innate Lymphoid Cells

PubMed Central

Scoville, Steven D.; Freud, Aharon G.; Caligiuri, Michael A.

2017-01-01

Decades after the discovery of natural killer (NK) cells, their developmental pathways in mice and humans have not yet been completely deciphered. Accumulating evidence indicates that NK cells can develop in multiple tissues throughout the body. Moreover, detailed and comprehensive models of NK cell development were proposed soon after the turn of the century. However, with the recent identification and characterization of other subtypes of innate lymphoid cells (ILCs), which show some overlapping functional and phenotypic features with NK cell developmental intermediates, the distinct stages through which human NK cells develop from early hematopoietic progenitor cells remain unclear. Thus, there is a need to reassess and refine older models of NK cell development in the context of new data and in the era of ILCs. Our group has focused on elucidating the developmental pathway of human NK cells in secondary lymphoid tissues (SLTs), including tonsils and lymph nodes. Here, we provide an update of recent progress that has been made with regard to human NK cell development in SLTs, and we discuss these new findings in the context of contemporary models of ILC development. PMID:28396671

Modeling Human Natural Killer Cell Development in the Era of Innate Lymphoid Cells.

PubMed

Scoville, Steven D; Freud, Aharon G; Caligiuri, Michael A

2017-01-01

Decades after the discovery of natural killer (NK) cells, their developmental pathways in mice and humans have not yet been completely deciphered. Accumulating evidence indicates that NK cells can develop in multiple tissues throughout the body. Moreover, detailed and comprehensive models of NK cell development were proposed soon after the turn of the century. However, with the recent identification and characterization of other subtypes of innate lymphoid cells (ILCs), which show some overlapping functional and phenotypic features with NK cell developmental intermediates, the distinct stages through which human NK cells develop from early hematopoietic progenitor cells remain unclear. Thus, there is a need to reassess and refine older models of NK cell development in the context of new data and in the era of ILCs. Our group has focused on elucidating the developmental pathway of human NK cells in secondary lymphoid tissues (SLTs), including tonsils and lymph nodes. Here, we provide an update of recent progress that has been made with regard to human NK cell development in SLTs, and we discuss these new findings in the context of contemporary models of ILC development.

Influenza Virus Directly Infects Human Natural Killer Cells and Induces Cell Apoptosis▿

PubMed Central

Mao, Huawei; Tu, Wenwei; Qin, Gang; Law, Helen Ka Wai; Sia, Sin Fun; Chan, Ping-Lung; Liu, Yinping; Lam, Kwok-Tai; Zheng, Jian; Peiris, Malik; Lau, Yu-Lung

2009-01-01

Influenza is an acute respiratory viral disease that is transmitted in the first few days of infection. Evasion of host innate immune defenses, including natural killer (NK) cells, is important for the virus's success as a pathogen of humans and other animals. NK cells encounter influenza viruses within the microenvironment of infected cells and are important for host innate immunity during influenza virus infection. It is therefore important to investigate the direct effects of influenza virus on NK cells. In this study, we demonstrated for the first time that influenza virus directly infects and replicates in primary human NK cells. Viral entry into NK cells was mediated by both clathrin- and caveolin-dependent endocytosis rather than through macropinocytosis and was dependent on the sialic acids on cell surfaces. In addition, influenza virus infection induced a marked apoptosis of NK cells. Our findings suggest that influenza virus can directly target and kill NK cells, a potential novel strategy of influenza virus to evade the NK cell innate immune defense that is likely to facilitate viral transmission and may also contribute to virus pathogenesis. PMID:19587043

Lifestyles and mental health status are associated with natural killer cell and lymphokine-activated killer cell activities.

PubMed

Morimoto, K; Takeshita, T; Inoue-Sakurai, C; Maruyama, S

2001-04-10

We investigated the association of lifestyle and mental health status with natural killer (NK) cell and lymphokine-activated killer (LAK) cell activities in healthy males. NK cell activity was determined in 105 male workers and LAK cell activity was determined in 54 male workers. Peripheral blood was obtained from each subject and peripheral blood mononuclear cells (PBMC) were isolated from the blood. These PBMC were used as effector cells. LAK cells were generated by incubation of PBMC with interleukin-2 for 72 h. NK cell activity against NK-sensitive K562 cells and LAK cell activity against NK-resistant Raji cells were examined by 51Cr release assay. Overall lifestyles were evaluated according to the answers on a questionnaire regarding eight health practices (cigarette smoking, alcohol consumption, eating breakfast, hours of sleep, hours of work, physical exercise, nutritional balance, mental stress). Subjects with a good overall lifestyle showed significantly higher NK cell (P < 0.05) and LAK cell (P < 0.05) activities than those with a poor overall lifestyles. Among eight lifestyle factors, cigarette smoking has relatively strong effects on NK cell and LAK cell activities. Subjects who complained of unstable mental status had significantly lower NK cell activity than those who reported stable mental status. When subjects were divided into four groups by lifestyle and mental health status, subjects who had poor or moderate lifestyle and reported unstable mental status showed the lowest NK cell activity and subjects who had good lifestyle and reported stable mental status showed the highest NK cell activity among four groups.

Leveraging natural killer cells for cancer immunotherapy.

PubMed

Grossenbacher, Steven K; Aguilar, Ethan G; Murphy, William J

2017-05-01

Natural killer (NK) cells are potent antitumor effector cells of the innate immune system. Based on their ability to eradicate tumors in vitro and in animal models, significant enthusiasm surrounds the prospect of leveraging human NK cells as vehicles for cancer immunotherapy. While interest in manipulating the effector functions of NK cells has existed for over 30 years, there is renewed optimism for this approach today. Although T cells receive much of the clinical and preclinical attention when it comes to cancer immunotherapy, new strategies are utilizing adoptive NK-cell immunotherapy and monoclonal antibodies and engineered molecules which have been developed to specifically activate NK cells against tumors. Despite the numerous challenges associated with the preclinical and clinical development of NK cell-based therapies for cancer, NK cells possess many unique immunological properties and hold the potential to provide an effective means for cancer immunotherapy.

NK cells interactions with dendritic cells shape innate and adaptive immunity.

PubMed

Brilot, Fabienne; Strowig, Till; Munz, Christian

2008-05-01

While natural killer (NK) cells received their name from their ability to mediate spontaneous cytotoxicity, it has recently become clear that they require activation to target most transformed and infected cells. Dendritic cells (DCs) have been shown to mediate NK cell activation during innate immune responses. Surprisingly, this interaction was recently reported to be required to restrict infections by NK cells, and to take place in secondary lymphoid organs. Here we review these recent studies on NK cell interactions with DCs, discuss the molecular mechanisms underlying the cross-talk between these two innate lymphocyte populations, and out-line how DCs and NK cells synergize to enhance innate immunity against microbes and tumors as well as shape the adaptive immune system. Based on this better understanding, we propose that NK cells should be targeted for their protective functions and as an adjuvant during immunotherapy development.

Cryopreservation has no effect on function of natural killer cells differentiated in vitro from umbilical cord blood CD34(+) cells.

PubMed

Domogala, Anna; Madrigal, J Alejandro; Saudemont, Aurore

2016-06-01

Natural killer (NK) cells offer the potential for a powerful cellular immunotherapy because they can target malignant cells without being direct effectors of graft-versus-host disease. We have previously shown that high numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34(+) cells. To develop a readily available, off-the-shelf cellular product, it is essential that NK cells differentiated in vitro can be frozen and thawed while maintaining the same phenotype and functions. We evaluated the phenotype and function of fresh and frozen NK cells differentiated in vitro. We also assessed whether the concentration of NK cells at the time of freezing had an impact on cell viability. We found that cell concentration of NK cells at the time of freezing did not have an impact on their viability and on cell recovery post-thaw. Moreover, freezing of differentiated NK cells in vitro did not affect their phenotype, cytotoxicity and degranulation capacity toward K562 cells, cytokine production and proliferation. We are therefore able to generate large numbers of functional NK cells from CB CD34(+) cells that maintain the same phenotype and function post-cryopreservation, which will allow for multiple infusions of a highly cytotoxic NK cell product. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

PubMed

Cho, Fu-Nan; Chang, Tsung-Hsien; Shu, Chih-Wen; Ko, Ming-Chin; Liao, Shuen-Kuei; Wu, Kang-Hsi; Yu, Ming-Sun; Lin, Shyh-Jer; Hong, Ying-Chung; Chen, Chien-Hsun; Hung, Chien-Hui; Chang, Yu-Hsiang

2014-01-01

Natural killer (NK) cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs), which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL) cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

Herpes simplex virus antigens directly activate NK cells via TLR2, thus facilitating their presentation to CD4 T lymphocytes.

PubMed

Kim, Min; Osborne, Naomi R; Zeng, Weiguang; Donaghy, Heather; McKinnon, Kay; Jackson, David C; Cunningham, Anthony L

2012-05-01

NK cells infiltrate human herpetic lesions, but their role has been underexplored. HSV can stimulate innate immune responses via surface TLR2, which is expressed on monocyte-derived dendritic cells (DCs) and NK cells. In this study, UV-inactivated HSV1/2 and immunodominant HSV2 glycoprotein D peptides conjugated to the TLR2 agonist dipalmitoyl-S-glyceryl cysteine stimulated CD4 T lymphocyte IFN-γ responses within PBMCs or in coculture with monocyte-derived DCs. NK cells contributed markedly to the PBMC responses. Furthermore, NK cells alone were activated directly by both Ags, also upregulating HLA-DR and HLA-DQ and then they activated autologous CD4 T lymphocytes. Using Transwells, Ag-stimulated NK cells and CD4 T lymphocytes were shown to interact through both cell-to-cell contact and cytokines, differing in relative importance in different donors. A distinct immunological synapse between Ag-stimulated NK cells and CD4 T lymphocytes was observed, indicating the significance of their cell-to-cell contact. A large proportion (57%) of NK cells was also in contact with CD4 T lymphocytes in the dermal infiltrate of human recurrent herpetic lesions. Thus, NK cells stimulated by TLR2-activating HSV Ags can present Ag alone or augment the role of DCs in vitro and perhaps in herpetic lesions or draining lymph nodes. In addition to DCs, NK cells should be considered as targets for adjuvants during HSV vaccine development.

Cytokine activation induces human memory-like NK cells.

PubMed

Romee, Rizwan; Schneider, Stephanie E; Leong, Jeffrey W; Chase, Julie M; Keppel, Catherine R; Sullivan, Ryan P; Cooper, Megan A; Fehniger, Todd A

2012-12-06

Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56(dim) NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.

“Multi-Omics” Analyses of the Development and Function of Natural Killer Cells

PubMed Central

Zhou, Yonggang; Xu, Xiuxiu; Tian, Zhigang; Wei, Haiming

2017-01-01

For over four decades, our understanding of natural killer (NK) cells has evolved from the original description of cluster of differentiation (CD)56+CD3− to establishing NK cells as an important subset of innate lymphocytes in the host’s surveillance against viral infections and malignancy. The progress of research on the fundamental properties and therapeutic prospects for translational medicine using NK cells excites immunologists and clinicians. Over the past decade, numerous advances in “-omics”-scale methods and new technological approaches have addressed many essential questions in the biology of NK cells. We now have further understanding of the overall molecular mechanisms of action that determine the development, function, plasticity, diversity, and immune reactivity of NK cells. These findings are summarized here, and our view on how to study NK cells using “multi-omics” is highlighted. We also describe “-omics” analyses of the relationships between NK cells and viral infection, tumorigenesis, and autoimmune diseases. Ultimately, a deeper and more comprehensive understanding of NK cells in multiple conditions will provide more effective strategies to manipulate NK cells for the treatment of human disease. PMID:28928751

Epstein-Barr virus-associated T/natural killer-cell lymphoproliferative disorders.

PubMed

Park, Sanghui; Ko, Young H

2014-01-01

Primary infection with Epstein-Barr virus (EBV) is usually asymptomatic and, in a normal host, EBV remains latent in B cells after primary infection for the remainder of life. Uncommonly, EBV can infect T or natural killer (NK) cells in a person with a defect in innate immunity, and EBV infection can cause unique systemic lymphoproliferative diseases (LPD) of childhood. Primary infection in young children can be complicated by hemophagocytic lymphohistiocytosis or fulminant systemic T-cell LPD of childhood. Uncommonly, patients can develop chronic active EBV (CAEBV) disease-type T/NK LPD, which includes CAEBV infection of the systemic form, hydroa vacciniforme-like T-cell LPD, and mosquito-bite hypersensitivity. The clinical course of CAEBV disease-type T/NK LPD can be smoldering, persistent or progressive, depending on the balance between viral factors and host immunity. Aggressive NK-cell leukemia, hydroa vacciniforme-like T-cell lymphoma, or uncommonly extranodal NK/T-cell lymphoma can develop in children and young adults with CAEBV disease-type T/NK-cell LPD. Extranodal T/NK-cell lymphoma is a disease of adults, and its incidence begins to increase in the third decade and comprises the major subtype of T/NK LPD throughout life. Aggressive NK-cell leukemia and nodal T/NK-cell lymphoma of the elderly are fulminant diseases, and immune senescence may be an important pathogenetic factor. This review describes the current progress in identifying different types of EBV-associated T/NK-cell LPD and includes a brief presentation of data from Korea. © 2014 Japanese Dermatological Association.

Natural Killer Cell Functions during the Innate Immune Response to Pathogenic Streptococci

PubMed Central

Lemire, Paul; Galbas, Tristan; Thibodeau, Jacques; Segura, Mariela

2017-01-01

Dendritic cells (DCs) and NK cells play a crucial role in the first phase of host defense against infections. Group B Streptococcus (GBS) and Streptococcus suis are encapsulated streptococci causing severe systemic inflammation, leading to septicemia and meningitis. Yet, the involvement of NK cells in the innate immune response to encapsulated bacterial infection is poorly characterized. Here, it was observed that these two streptococcal species rapidly induce the release of IFN-γ and that NK cells are the major cell type responsible for this production during the acute phase of the infection. Albeit S. suis capacity to activate NK cells was lower than that of GBS, these cells partially contribute to S. suis systemic infection; mainly through amplification of the inflammatory loop. In contrast, such a role was not observed during GBS systemic infection. IFN-γ release by NK cells required the presence of DCs, which in turn had a synergistic effect on DC cytokine production. These responses were mainly mediated by direct DC-NK cell contact and partially dependent on soluble factors. Though IL-12 and LFA-1 were shown to be critical in S. suis-mediated activation of the DC-NK cell crosstalk, different or redundant molecular pathways modulate DC-NK interactions during GBS infection. The bacterial capsular polysaccharides also differently modulated NK cell activation. Together, these results demonstrated a role of NK cells in the innate immune response against encapsulated streptococcal infections; yet the molecular pathways governing NK activation seem to differ upon the pathogen and should not be generalized when studying bacterial infections. PMID:28706510

A Signal Peptide Derived from hsp60 Binds HLA-E and Interferes with CD94/NKG2A Recognition

PubMed Central

Michaëlsson, Jakob; Teixeira de Matos, Cristina; Achour, Adnane; Lanier, Lewis L.; Kärre, Klas; Söderström, Kalle

2002-01-01

Human histocompatibility leukocyte antigen (HLA)-E is a nonclassical major histocompatibility complex (MHC) class I molecule which presents a restricted set of nonameric peptides, derived mainly from the signal sequence of other MHC class I molecules. It interacts with CD94/NKG2 receptors expressed on the surface of natural killer (NK) cells and T cell subsets. Here we demonstrate that HLA-E also presents a peptide derived from the leader sequence of human heat shock protein 60 (hsp60). This peptide gains access to HLA-E intracellularly, resulting in up-regulated HLA-E/hsp60 signal peptide cell-surface levels on stressed cells. Notably, HLA-E molecules in complex with the hsp60 signal peptide are no longer recognized by CD94/NKG2A inhibitory receptors. Thus, during cellular stress an increased proportion of HLA-E molecules may bind the nonprotective hsp60 signal peptide, leading to a reduced capacity to inhibit a major NK cell population. Such stress induced peptide interference would gradually uncouple CD94/NKG2A inhibitory recognition and provide a mechanism for NK cells to detect stressed cells in a peptide-dependent manner. PMID:12461076

HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells

PubMed Central

Gornalusse, Germán G.; Hirata, Roli K.; Funk, Sarah; Riolobos, Laura; Lopes, Vanda S.; Manske, Gabriel; Prunkard, Donna; Colunga, Aric G.; Hanafi, Laïla-Aïcha; Clegg, Dennis O.; Turtle, Cameron; Russell, David W.

2017-01-01

Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this ‘missing self’ response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8+ T cells, do not bind anti-HLA antibodies, and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression. PMID:28504668

HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells.

PubMed

Gornalusse, Germán G; Hirata, Roli K; Funk, Sarah E; Riolobos, Laura; Lopes, Vanda S; Manske, Gabriel; Prunkard, Donna; Colunga, Aric G; Hanafi, Laïla-Aïcha; Clegg, Dennis O; Turtle, Cameron; Russell, David W

2017-08-01

Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8 + T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.

Ligation of CD8α on human natural killer cells prevents activation-induced apoptosis and enhances cytolytic activity

PubMed Central

Addison, Elena G; North, Janet; Bakhsh, Ismail; Marden, Chloe; Haq, Sumaira; Al-Sarraj, Samia; Malayeri, Reza; Wickremasinghe, R Gitendra; Davies, Jeffrey K; Lowdell, Mark W

2005-01-01

It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric α/α form are more cytotoxic than their CD8– counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8– NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8α+ cells after lysis of the same targets. We report that cross-linking of the CD8α chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK-cell apoptosis from which CD8α+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK-cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti-MHC Class I antibody. Thus, in contrast to the CD8– subset, CD8α+ NK cells are capable of sequential lysis of multiple target cells. PMID:16236125

Involvement of NK cells against tumors and parasites.

PubMed

Papazahariadou, M; Athanasiadis, G I; Papadopoulos, E; Symeonidou, I; Hatzistilianou, M; Castellani, M L; Bhattacharya, K; Shanmugham, L N; Conti, P; Frydas, S

2007-01-01

Host resistance against pathogens depends on a complex interplay of innate and adaptive immune mechanisms. Acting as an early line of defence, the immune system includes activation of neutrophils, tissue macrophages, monocytes, dendritic cells, eosinophils and natural killer (NK) cells. NK cells are lymphoid cells that can be activated without previous stimulation and are therefore like macrophages in the first line of defence against tumor cells and a diverse range of pathogens. NK cells mediate significant activity and produce high levels of proinflammatory cytokines in response to infection. Their cytotoxicity production is induced principally by monocyte-, macrophage- and dendritic cell-derived cytokines, but their activation is also believed to be cytokine-mediated. Recognition of infection by NK cells is accomplished by numerous activating and inhibitory receptors on the NK cells' surface that selectively trigger the cytolytic activity in a major histocompability complex-independent manner. NK cells have trypanocidal activity of fibroblast cells and mediate direct destruction of extracellular epimastigote and trypomastigote forms of T. cruzi and T. lewisi in vitro; moreover, they kill plasmodia-infected erythrocytes directly through cell-cell interaction. This review provides a more detailed analysis of how NK cells recognize and respond to parasites and how they mediate cytotoxicity against tumor cells. Also the unique role of NK cells in innate immunity to infection and the relationship between parasites and carcinogenesis are discussed.

Is There Natural Killer Cell Memory and Can It Be Harnessed by Vaccination? Natural Killer Cells in Vaccination.

PubMed

Neely, Harold R; Mazo, Irina B; Gerlach, Carmen; von Andrian, Ulrich H

2017-12-18

Natural killer (NK) cells have historically been considered to be a part of the innate immune system, exerting a rapid response against pathogens and tumors in an antigen (Ag)-independent manner. However, over the past decade, evidence has accumulated suggesting that at least some NK cells display certain characteristics of adaptive immune cells. Indeed, NK cells can learn and remember encounters with a variety of Ags, including chemical haptens and viruses. Upon rechallenge, memory NK cells mount potent recall responses selectively to those Ags. This phenomenon, traditionally termed "immunological memory," has been reported in mice, nonhuman primates, and even humans and appears to be concentrated in discrete NK cell subsets. Because immunological memory protects against recurrent infections and is the central goal of active vaccination, it is crucial to define the mechanisms and consequences of NK cell memory. Here, we summarize the different kinds of memory responses that have been attributed to specific NK cell subsets and discuss the possibility to harness NK cell memory for vaccination purposes. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

Complementary Phosphorylation Sites in the Adaptor Protein SLP-76 Promote Synergistic Activation of Natural Killer Cells

PubMed Central

Kim, Hun Sik; Long, Eric O.

2013-01-01

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require the induction of synergistic signals from co-activation receptors, such as CD314 (NKG2D) and CD244 (2B4), which bind to ligands expressed on target cells. Synergy is required to overcome inhibition of the guanine nucleotide exchange factor (GEF) Vav1, a central regulator of NK cell activation, by the E3 ubiquitin ligase c-Cbl. However, the molecular basis for this synergy is unknown. Here, we showed that the adaptor protein Src homology 2 (SH2) domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) was required for this synergy, and that distinct tyrosine residues in SLP-76 were phosphorylated by each receptor of a synergistic pair. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76, each of which enables binding of SLP-76 to Vav1, was unique to receptors that stimulate ligand-dependent target cell killing, because antibody-dependent stimulation by Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that complementation of separate phospho-tyrosine targets in SLP-76 forms the basis of synergistic NK cell activation. PMID:22786724

The use of microRNA by human viruses: lessons from NK cells and HCMV infection.

PubMed

Goldberger, Tal; Mandelboim, Ofer

2014-11-01

Depending on ethnicity and on social conditions, between 40 and 90 % of the population is infected with human cytomegalovirus (HCMV). In immunocompetent patients, the virus may cause an acute disease and then revert to a state of latency, which enables its coexistence with the human host. However, in cases of immunosuppression or in neonatal infections, HCMV can cause serious long-lasting illnesses. HCMV has developed multiple mechanisms in order to escape its elimination by the immune system, specifically by two killer cell types of the adaptive and the innate immune systems; cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, respectively. Another fascinating aspect of HCMV is that like other highly developed herpesviruses, it expresses its own unique set of microRNAs. Here, we initially describe how the activity of NK cells is regulated under normal conditions and during infection. Then, we discuss what is currently known about HCMV microRNA-mediated interactions, with special emphasis on immune modulation and NK cell evasion. We further illustrate the significant modulation of cellular microRNAs during HCMV infection. Although, the full target spectrum of HCMV microRNAs is far from being completely elucidated, it can already be concluded that HCMV uses its "multitasking" microRNAs to globally affect its own life cycle, as well as important cellular and immune-related pathways.

Efficient mRNA-Based Genetic Engineering of Human NK Cells with High-Affinity CD16 and CCR7 Augments Rituximab-Induced ADCC against Lymphoma and Targets NK Cell Migration toward the Lymph Node-Associated Chemokine CCL19.

PubMed

Carlsten, Mattias; Levy, Emily; Karambelkar, Amrita; Li, Linhong; Reger, Robert; Berg, Maria; Peshwa, Madhusudan V; Childs, Richard W

2016-01-01

For more than a decade, investigators have pursued methods to genetically engineer natural killer (NK) cells for use in clinical therapy against cancer. Despite considerable advances in viral transduction of hematopoietic stem cells and T cells, transduction efficiencies for NK cells have remained disappointingly low. Here, we show that NK cells can be genetically reprogramed efficiently using a cGMP-compliant mRNA electroporation method that induces rapid and reproducible transgene expression in nearly all transfected cells, without negatively influencing their viability, phenotype, and cytotoxic function. To study its potential therapeutic application, we used this approach to improve key aspects involved in efficient lymphoma targeting by adoptively infused ex vivo-expanded NK cells. Electroporation of NK cells with mRNA coding for the chemokine receptor CCR7 significantly promoted migration toward the lymph node-associated chemokine CCL19. Further, introduction of mRNA coding for the high-affinity antibody-binding receptor CD16 (CD16-158V) substantially augmented NK cell cytotoxicity against rituximab-coated lymphoma cells. Based on these data, we conclude that this approach can be utilized to genetically modify multiple modalities of NK cells in a highly efficient manner with the potential to improve multiple facets of their in vivo tumor targeting, thus, opening a new arena for the development of more efficacious adoptive NK cell-based cancer immunotherapies.

Bridging innate NK cell functions with adaptive immunity.

PubMed

Marcenaro, Emanuela; Carlomagno, Simona; Pesce, Silvia; Moretta, Alessandro; Sivori, Simona

2011-01-01

Killer Ig-like receptors (KIRs) are major human NK receptors displaying either inhibitory or activating functions which recognize allotypic determinants of HLA-class I molecules. Surprisingly, NK cell treatment with CpG-ODN (TLR9 ligands) results in selective down-modulation of KIR3DL2, its co-internalization with CpG-ODN and its translocation to TLR9-rich early endosomes. This novel KIR-associated function may offer clues to better understand the possible role of certain KIRs and also emphasizes the involvement of NK cells in the course of microbial infections. NK cells are involved not only in innate immune responses against viruses and tumors but also participate in the complex network of cell-to cell interaction that leads to the development of adaptive immune responses. In this context the interaction of NK cells with DC appears to play a crucial role in the acquisition of CCR7, a chemokine receptor that enables NK cells to migrate towards lymph nodes in response to CCL19 and/or CCL21. Analysis of NK cell clones revealed that KIR-mismatched but not KIR-matched NK cells acquire CCR7. These data have important implications in haploidentical haematopoietic stem cell transplantation (HSCT), in which KIR-mismatched NK cells may acquire the ability to migrate to secondary lymphoid compartments (SLCs), where they can kill recipient's antigen presenting cells (APCs) and T cells thus preventing graft versus host (and host vs. graft) reactions.

CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells.

PubMed

Han, Jianfeng; Chu, Jianhong; Keung Chan, Wing; Zhang, Jianying; Wang, Youwei; Cohen, Justus B; Victor, Aaron; Meisen, Walter H; Kim, Sung-hak; Grandi, Paola; Wang, Qi-En; He, Xiaoming; Nakano, Ichiro; Chiocca, E Antonio; Glorioso, Joseph C; Kaur, Balveen; Caligiuri, Michael A; Yu, Jianhua

2015-07-09

Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.

Voluntary Running Suppresses Tumor Growth through Epinephrine- and IL-6-Dependent NK Cell Mobilization and Redistribution.

PubMed

Pedersen, Line; Idorn, Manja; Olofsson, Gitte H; Lauenborg, Britt; Nookaew, Intawat; Hansen, Rasmus Hvass; Johannesen, Helle Hjorth; Becker, Jürgen C; Pedersen, Katrine S; Dethlefsen, Christine; Nielsen, Jens; Gehl, Julie; Pedersen, Bente K; Thor Straten, Per; Hojman, Pernille

2016-03-08

Regular exercise reduces the risk of cancer and disease recurrence. Yet the mechanisms behind this protection remain to be elucidated. In this study, tumor-bearing mice randomized to voluntary wheel running showed over 60% reduction in tumor incidence and growth across five different tumor models. Microarray analysis revealed training-induced upregulation of pathways associated with immune function. NK cell infiltration was significantly increased in tumors from running mice, whereas depletion of NK cells enhanced tumor growth and blunted the beneficial effects of exercise. Mechanistic analyses showed that NK cells were mobilized by epinephrine, and blockade of β-adrenergic signaling blunted training-dependent tumor inhibition. Moreover, epinephrine induced a selective mobilization of IL-6-sensitive NK cells, and IL-6-blocking antibodies blunted training-induced tumor suppression, intratumoral NK cell infiltration, and NK cell activation. Together, these results link exercise, epinephrine, and IL-6 to NK cell mobilization and redistribution, and ultimately to control of tumor growth. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of Neurokinin 3 Receptor Signaling in Oral Squamous Cell Carcinoma.

PubMed

Obata, Kyoichi; Shimo, Tsuyoshi; Okui, Tatsuo; Matsumoto, Kenichi; Takada, Hiroyuki; Takabatake, Kiyofumi; Kunisada, Yuki; Ibaragi, Soichiro; Yoshioka, Norie; Kishimoto, Koji; Nagatsuka, Hitoshi; Sasaki, Akira

2017-11-01

The neurokinin 3 receptor (NK-3R) is differentially expressed in the central nervous system including cases of human oral squamous cell carcinoma. However, the role of NK-3R signaling in oral squamous cell carcinoma is not well known. NK-3R expression in surgically resected oral squamous cell carcinoma was examined immunohistochemically and the strength of the expression was quantified. We evaluated the function of NK-3R signaling using NK-3R antagonist in human oral squamous cell carcinoma bone invasion mouse model. NK-3R was significantly expressed in tumor cells that had invaded the bone matrix compared to the oral side tumor cells. SB222200, a selective antagonist of NK-3R, significantly suppressed the radiographic osteolytic lesion and tumorigenesis. NK-3R signaling is a potential target for the treatment of oral squamous cell carcinoma in cases of bone destruction. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Rhizopus oryzae hyphae are damaged by human natural killer (NK) cells, but suppress NK cell mediated immunity.

PubMed

Schmidt, Stanislaw; Tramsen, Lars; Perkhofer, Susanne; Lass-Flörl, Cornelia; Hanisch, Mitra; Röger, Frauke; Klingebiel, Thomas; Koehl, Ulrike; Lehrnbecher, Thomas

2013-07-01

Mucormycosis has a high mortality and is increasingly diagnosed in hematopoietic stem cell transplant (HSCT) recipients. In this setting, there is a growing interest to restore host defense to combat infections by adoptively transferring donor-derived immunocompetent cells. Natural killer (NK) cells exhibit antitumor and antiinfective activity, but the interaction with Mucormycetes is unknown. Our data demonstrate that both unstimulated and IL-2 prestimulated human NK cells damage Rhizopus oryzae hyphae, but do not affect resting conidia. The damage of the fungus is mediated, at least in part, by perforin. R. oryzae hyphae decrease the secretion of immunoregulatory molecules by NK cells, such as IFN-γ and RANTES, indicating an immunosuppressive effect of the fungus. Our data indicate that NK cells exhibit activity against Mucormycetes and future research should evaluate NK cells as a potential tool for adoptive immunotherapy in HSCT. Copyright © 2012 Elsevier GmbH. All rights reserved.

A King Bolete, Boletus edulis (Agaricomycetes), RNA Fraction Stimulates Proliferation and Cytotoxicity of Natural Killer Cells Against Myelogenous Leukemia Cells.

PubMed

Lemieszek, Marta Kinga; Nunes, Fernando Herminio Ferreira Milheiro; Sawa-Wejksza, Katarzyna; Rzeski, Wojciech

2017-01-01

Numerous studies indicate the crucial role of natural killer (NK) cells in the prevention of tumor growth and inhibition of their metastasis, which suggests the possibility of their use in cancer treatment. This therapeutic strategy required finding a selective NK cell stimulator that, upon administration, did not disturb organism homeostasis, unlike natural activators (interleukin-2 or interleukin-12). Because the majority of anticancer agents derived from Basidiomycetes are able to stimulate lymphocytes, we describe the influence of Boletus edulis RNA on a human NK cell line (NK92). Our studies showed that a B. edulis RNA fraction was not toxic against NK92 cells. Furthermore, the tested fraction significantly stimulated NK92 cell proliferation and their cytotoxicity against tumor cells. We demonstrate here, to our knowledge for the first time, that B. edulis RNA enhances NK cell activity and possesses immunomodulatory potential.

Zika Virus Escapes NK Cell Detection by Upregulating Major Histocompatibility Complex Class I Molecules.

PubMed

Glasner, Ariella; Oiknine-Djian, Esther; Weisblum, Yiska; Diab, Mohammad; Panet, Amos; Wolf, Dana G; Mandelboim, Ofer

2017-11-15

NK cells are innate lymphocytes that participate in many immune processes encompassing cancer, bacterial and fungal infection, autoimmunity, and even pregnancy and that specialize in antiviral defense. NK cells express inhibitory and activating receptors and kill their targets when activating signals overpower inhibitory signals. The NK cell inhibitory receptors include a uniquely diverse array of proteins named killer cell immunoglobulin-like receptors (KIRs), the CD94 family, and the leukocyte immunoglobulin-like receptor (LIR) family. The NK cell inhibitory receptors recognize mostly major histocompatibility complex (MHC) class I (MHC-I) proteins. Zika virus has recently emerged as a major threat due to its association with birth defects and its pandemic potential. How Zika virus interacts with the immune system, and especially with NK cells, is unclear. Here we show that Zika virus infection is barely sensed by NK cells, since little or no increase in the expression of activating NK cell ligands was observed following Zika infection. In contrast, we demonstrate that Zika virus infection leads to the upregulation of MHC class I proteins and consequently to the inhibition of NK cell killing. Mechanistically, we show that MHC class I proteins are upregulated via the RIGI-IRF3 pathway and that this upregulation is mediated via beta interferon (IFN-β). Potentially, countering MHC class I upregulation during Zika virus infection could be used as a prophylactic treatment against Zika virus. IMPORTANCE NK cells are innate lymphocytes that recognize and eliminate various pathogens and are known mostly for their role in controlling viral infections. NK cells express inhibitory and activating receptors, and they kill or spare their targets based on the integration of inhibitory and activating signals. Zika virus has recently emerged as a major threat to humans due to its pandemic potential and its association with birth defects. The role of NK cells in Zika virus infection is largely unknown. Here we demonstrate that Zika virus infection is almost undetected by NK cells, as evidenced by the fact that the expression of activating ligands for NK cells is not induced following Zika infection. We identified a mechanism whereby Zika virus sensing via the RIGI-IRF3 pathway resulted in IFN-β-mediated upregulation of MHC-I molecules and inhibition of NK cell activity. Countering MHC class I upregulation and boosting NK cell activity may be employed as prophylactic measures to combat Zika virus infection. Copyright © 2017 American Society for Microbiology.

Zika Virus Escapes NK Cell Detection by Upregulating Major Histocompatibility Complex Class I Molecules

PubMed Central

Glasner, Ariella; Oiknine-Djian, Esther; Weisblum, Yiska; Diab, Mohammad; Panet, Amos; Wolf, Dana G.

2017-01-01

ABSTRACT NK cells are innate lymphocytes that participate in many immune processes encompassing cancer, bacterial and fungal infection, autoimmunity, and even pregnancy and that specialize in antiviral defense. NK cells express inhibitory and activating receptors and kill their targets when activating signals overpower inhibitory signals. The NK cell inhibitory receptors include a uniquely diverse array of proteins named killer cell immunoglobulin-like receptors (KIRs), the CD94 family, and the leukocyte immunoglobulin-like receptor (LIR) family. The NK cell inhibitory receptors recognize mostly major histocompatibility complex (MHC) class I (MHC-I) proteins. Zika virus has recently emerged as a major threat due to its association with birth defects and its pandemic potential. How Zika virus interacts with the immune system, and especially with NK cells, is unclear. Here we show that Zika virus infection is barely sensed by NK cells, since little or no increase in the expression of activating NK cell ligands was observed following Zika infection. In contrast, we demonstrate that Zika virus infection leads to the upregulation of MHC class I proteins and consequently to the inhibition of NK cell killing. Mechanistically, we show that MHC class I proteins are upregulated via the RIGI-IRF3 pathway and that this upregulation is mediated via beta interferon (IFN-β). Potentially, countering MHC class I upregulation during Zika virus infection could be used as a prophylactic treatment against Zika virus. IMPORTANCE NK cells are innate lymphocytes that recognize and eliminate various pathogens and are known mostly for their role in controlling viral infections. NK cells express inhibitory and activating receptors, and they kill or spare their targets based on the integration of inhibitory and activating signals. Zika virus has recently emerged as a major threat to humans due to its pandemic potential and its association with birth defects. The role of NK cells in Zika virus infection is largely unknown. Here we demonstrate that Zika virus infection is almost undetected by NK cells, as evidenced by the fact that the expression of activating ligands for NK cells is not induced following Zika infection. We identified a mechanism whereby Zika virus sensing via the RIGI-IRF3 pathway resulted in IFN-β-mediated upregulation of MHC-I molecules and inhibition of NK cell activity. Countering MHC class I upregulation and boosting NK cell activity may be employed as prophylactic measures to combat Zika virus infection. PMID:28878071

The expression of human natural killer cell receptors in early life.

PubMed

Sundström, Y; Nilsson, C; Lilja, G; Kärre, K; Troye-Blomberg, M; Berg, L

2007-01-01

Natural killer (NK) cells play an important role in tumour immunosurveillance and the early defence against viral infections. Recognition of altered cells (i.e. infected- or tumour-cells) is achieved through a multiple receptor recognition strategy which gives the NK cells inhibitory or activating signals depending on the ligands present on the target cell. NK cells originate from the bone marrow where they develop and proliferate. However, further maturation processes and homeostasis of NK cells in peripheral blood are not well understood. To determine the proportions of cells and the expression of NK cell receptors, mononuclear cells from children at three time points during early childhood were compared, i.e. cord blood (CB), 2 and 5 years of age. The proportion of NK cells was high in CB, but the interferon-gamma (IFN-gamma) production low compared to later in life. In contrast, the proportion of T cells was low in CB. This may indicate a deviation of the regulatory function of NK cells in CB compared to later in life, implying an importance of innate immunity in early life before the adaptive immune system matures. Additionally, we found that the proportion of LIR-1(+) NK cells increased with increasing age while CD94(+)NKG2C(-) (NKG2A(+)) NK cells and the level of expression of NKG2D, NKp30 and NKp46 decreased with age. These age related changes in NK cell populations defined by the expression of activating and inhibitory receptors may be the result of pathogen exposure and/or a continuation of the maturation process that begins in the bone marrow.

Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy

NASA Astrophysics Data System (ADS)

Taik Lim, Yong; Cho, Mi Young; Noh, Young-Woock; Chung, Jin Woong; Chung, Bong Hyun

2009-11-01

This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN- γ) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

Broadly targeted CD8 + T cell responses restricted by major histocompatibility complex E

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hansen, Scott G.; Wu, Helen L.; Burwits, Benjamin J.

Major histocompatibility complex (MHC)-E is a highly conserved, ubiquitously expressed, nonclassical, MHC-Ib molecule with limited polymorphism primarily involved in regulation of NK cell reactivity via interaction with NKG2/CD94 receptors. We found that vaccination of rhesus macaques with Rh157.5/.4 gene-deleted rhesus Cytomegalovirus (RhCMV) vectors uniquely diverts MHC-E function to presentation of highly diverse peptide epitopes to CD8α/β + T cells, approximately 4 distinct epitopes per 100 amino acids, in all tested protein antigens. Computational structural analysis revealed that a relatively stable, open binding groove in MHC-E attains broad peptide binding specificity by imposing a similar backbone configuration on bound peptides withmore » few restrictions based on amino acid side chains. Since MHC-E is up-regulated on cells infected with HIV/SIV and other persistent viruses to evade NK cell activity, MHC-E-restricted CD8 + T cell responses have the potential to exploit pathogen immune evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.« less

Broadly targeted CD8 + T cell responses restricted by major histocompatibility complex E

DOE PAGES

Hansen, Scott G.; Wu, Helen L.; Burwits, Benjamin J.; ...

2016-02-12

Major histocompatibility complex (MHC)-E is a highly conserved, ubiquitously expressed, nonclassical, MHC-Ib molecule with limited polymorphism primarily involved in regulation of NK cell reactivity via interaction with NKG2/CD94 receptors. We found that vaccination of rhesus macaques with Rh157.5/.4 gene-deleted rhesus Cytomegalovirus (RhCMV) vectors uniquely diverts MHC-E function to presentation of highly diverse peptide epitopes to CD8α/β + T cells, approximately 4 distinct epitopes per 100 amino acids, in all tested protein antigens. Computational structural analysis revealed that a relatively stable, open binding groove in MHC-E attains broad peptide binding specificity by imposing a similar backbone configuration on bound peptides withmore » few restrictions based on amino acid side chains. Since MHC-E is up-regulated on cells infected with HIV/SIV and other persistent viruses to evade NK cell activity, MHC-E-restricted CD8 + T cell responses have the potential to exploit pathogen immune evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.« less

NK cell recruitment and exercise: Potential immunotherapeutic role of shear stress and endothelial health.

PubMed

Evans, William

2017-11-01

Positive cancer patient outcomes, including increased time to recurrent events, have been associated with increased counts and function of natural killer (NK) cells. NK cell counts and function are elevated following acute exercise, and the generally accepted mechanism of increased recruitment suggests that binding of epinephrine releases NK cells from endothelial tissue via decreases in adhesion molecules following. I propose that blood flow-induced shear stress may also play a role in NK cell recruitment from the endothelium. Additionally, shear stress may play a role in improving NK cell function by decreasing oxidative stress. The relationship between shear stress and NK cell count and function can be tested by utilizing exercise and local heating with cuff inflation. If shear stress does play an important role, NK cell count and function will be improved in the non-cuffed exercise group, but not the cuffed limb. This paper will explore the mechanisms potentially explaining exercise-induced improvements in NK cell count and function, and propose a model for investigating these mechanisms. This mechanistic insight could aid in providing a novel, safe, relatively inexpensive, and non-invasive target for immunotherapy in cancer patients. Copyright © 2017. Published by Elsevier Ltd.

Plasmodium falciparum-Infected Erythrocytes Induce Granzyme B by NK Cells through Expression of Host-Hsp70

PubMed Central

Böttger, Evelyn; Multhoff, Gabriele; Kun, Jürgen F. J.; Esen, Meral

2012-01-01

In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo. PMID:22438997

Plasmodium falciparum-infected erythrocytes induce granzyme B by NK cells through expression of host-Hsp70.

PubMed

Böttger, Evelyn; Multhoff, Gabriele; Kun, Jürgen F J; Esen, Meral

2012-01-01

In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo.

Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites

PubMed Central

Messlinger, Helena; Sebald, Heidi; Heger, Lukas; Dudziak, Diana; Bogdan, Christian; Schleicher, Ulrike

2018-01-01

Activated natural killer (NK) cells release interferon (IFN)-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani). When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis) were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL)-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs) of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells reduced the expression of CD56 mRNA and protein on NK cells. We conclude that Leishmania activate NK cells via trans-presentation of IL-18 by monocytes and by a monocyte-derived soluble factor. IL-12 is needed to elicit the IFN-γ-response of NK cells, which is likely to be an important component of the innate control of the parasite. PMID:29472914

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rowe, Alexander M.; Brundage, Kathleen M.; Center for Immunopathology and Microbial Pathogenesis, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506

2007-06-01

The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesizedmore » that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.« less

Effects of tachykinins on uterine smooth muscle.

PubMed

Patak, E N; Pennefather, J N; Story, M E

2000-11-01

1. Sensory nerves supplying the mammalian uterus have been shown to contain substance P (SP) and neurokinin (NK)A. This review presents some of the advances that have led to a greater understanding of the effects of tachykinins on uterine smooth muscle. 2. The cell-surface peptidase neprilysin (EC.3 24.11, endopeptidase 24.11, enkephalinase, CALLA, CD10) has been shown to play a major role in regulating the actions of tachykinins on both rat and human myometrium. Because this peptidase is known to be regulated by steroids and pregnancy, its effects may be of physiological relevance. 3. Tachykinins produce contractions of isolated myometrial preparations from non-pregnant rats and mice. The NK2 receptor mediates these effects in rat uterus, while the NK1 receptor may mediate these effects in the mouse uterus. 4. The effects of tachykinins have been examined on myometrial preparations obtained at Caesarean section from near-term pregnant women. In the presence of the peptidase inhibitors (thiorphan, captopril and bestatin), the mammalian tachykinins SP, NKA and NKB produced concentration-dependent uterine contractions. 5. The order of agonist potency NKA > SP = NKB suggested that NK2 receptors mediate uterine contractions in the human. This was confirmed using the stable analogues [Sar9,Met(O2)11]SP, [Lys5MeLeu9Nle10]NKA(4-10) and [N-MePhe7]NKB, which are NK1, NK2 and NK3 receptor selective, respectively. Only [Lys5MeLeu9Nle10]NKA(4-10) produced concentration-related contractions of human uterine smooth muscle. 6. The experimental findings described in the present review, taken together with results published previously in the literature, indicate that tachykinin peptides may play a physiological or pathophysiological role in regulating uterine smooth muscle activity. However, more extensive research will be required to confirm such a role for these peptides.

An Hsp70 peptide initiates NK cell killing of leukemic blasts after stem cell transplantation.

PubMed

Gross, Catharina; Holler, Ernst; Stangl, Stefan; Dickinson, Anne; Pockley, A Graham; Asea, Alexzander A; Mallappa, Nagaraja; Multhoff, Gabriele

2008-04-01

In contrast to solid tumors, leukemic blasts frequently present both Hsp70 and HLA-E on their cell surface and thereby present activating and inhibitory signals to CD94(+) NK cells. In the first 12 months after stem cell transplantation (SCT) CD94(+) NK cells clearly dominate over CD3(+)/CD16(-)/56(-) T and CD3(+)/CD16(+)/56(+) NK-like T cells. An incubation of post-SCT-derived peripheral blood lymphocytes with the Hsp70 peptide TKD and IL-15 enhances the cell surface density of CD56/CD94 and initiates the cytolytic activity of NK cells against Hsp70/HLA-E double-positive autologous and allogeneic leukemic blasts. Hsp70 was identified as the target structure for TKD-activated NK cells.

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. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bacterial DNA-induced NK cell IFN-gamma production is dependent on macrophage secretion of IL-12.

PubMed

Chace, J H; Hooker, N A; Mildenstein, K L; Krieg, A M; Cowdery, J S

1997-08-01

Bacterial DNA (bDNA) activates B cells and macrophages and can augment inflammatory responses by inducing release of proinflammatory cytokines. We found that bDNA stimulation of mouse spleen cells induced NK cell IFN-gamma production that was dependent upon the presence of unmethylated CpG motifs, and oligonucleotides with internal CpG motifs could also induce splenocytes to secrete IFN-gamma. The bDNA-induced IFN-gamma response was strictly macrophages dependent. While splenocytes from SCID mice secreted IFN-gamma in response to bDNA, depletion of macrophages eliminated this response. Additionally, purified NK cells did not respond to bDNA; however, addition of macrophages restored the NK cell IFN-gamma response. Coculture of NK cells with preactivated macrophages further increased bDNA-induced NK cell IFN-gamma production. Anti-IL-12 or IL-10 inhibited bDNA-induced IFN-gamma response. Treatment of purified macrophages with bDNA resulted in IL-12 secretion accompanied by an increase in IL-12 p40 mRNA level. Although isolated NK cells did not make IFN-gamma in response to bDNA, NK cells costimulated with IL-12 gained the ability to respond to bDNA. These experiments show that bDNA induces macrophage IL-12 production which, in turn, stimulates NK cell IFN-gamma production. Macrophage-derived IL-12 renders NK cells responsive to bDNA permitting an even greater IFN-gamma response to bDNA.

Glutathione diminishes Dibutyltin- and tributyltin-induced loss of lytic function in human natural killer cells

PubMed Central

Powell, Jeralyn J.; Davis, McLisa V.; Whalen, Margaret M.

2008-01-01

This study investigated whether reduced glutathione (GSH) was able to alter the negative effects of tributyltin (TBT) or dibutyltin (DBT) on the lytic function of human natural killer (NK) cells. NK cells are an intital immune defense against the development of tumors or viral infections. TBT and DBT are widespread environmental contaminants, due to their various industrial applications. Both TBT and DBT have been shown to decrease the ability of NK cells to lyse tumor cells (lytic function). The results indicated that the presence of GSH during exposure of NK cells to TBT or DBT diminished the negative effect of the BT on the lytic function of NK cells. This suggests that interaction TBT and DBT with functionally relevant sulfhydryl groups in NK cells may be part of the mechanism by which they decrease NK lytic function. PMID:18821099

Glutathione diminishes tributyltin- and dibutyltin-induced loss of lytic function in human natural killer cells.

PubMed

Powell, Jeralyn J; Davis, McLisa V; Whalen, Margaret M

2009-01-01

This study investigated whether reduced glutathione (GSH) was able to alter the negative effects of tributyltin (TBT) or dibutyltin (DBT) on the lytic function of human natural killer (NK) cells. NK cells are an initial immune defense against the development of tumors or viral infections. TBT and DBT are widespread environmental contaminants, due to their various industrial applications. Both TBT and DBT have been shown to decrease the ability of NK cells to lyse tumor cells (lytic function). The results indicated that the presence of GSH during the exposure of NK cells to TBT or DBT diminished the negative effect of the butyltin on the lytic function of NK cells. This suggests that the interaction of TBT and DBT with functionally relevant sulfhydryl groups in NK cells may be part of the mechanism by which they decrease NK lytic function.

A High-Throughput Assay for Screening of Natural Products that Enhanced Tumoricidal Activity of NK Cells.

PubMed

Gong, Chenyuan; Ni, Zhongya; Yao, Chao; Zhu, Xiaowen; Ni, Lulu; Wang, Lixin; Zhu, Shiguo

2015-01-01

Recently, immunotherapy has shown a lot of promise in cancer treatment and different immune cell types are involved in this endeavor. Among different immune cell populations, NK cells are also an important component in unleashing the therapeutic activity of immune cells. Therefore, in order to enhance the tumoricidal activity of NK cells, identification of new small-molecule natural products is important. Despite the availability of different screening methods for identification of natural products, a simple, economic and high-throughput method is lacking. Hence, in this study, we have developed a high-throughput assay for screening and indentifying natural products that can enhance NK cell-mediated killing of cancer cells. We expanded human NK cell population from human peripheral blood mononuclear cells (PBMCs) by culturing these PBMCs with membrane-bound IL-21 and CD137L engineered K562 cells. Next, expanded NK cells were co-cultured with non-small cell lung cancer (NSCLC) cells with or without natural products and after 24 h of co-culturing, harvested supernatants were analyzed for IFN-γ secretions by ELISA method. We screened 502 natural products and identified that 28 candidates has the potential to induce IFN-γ secretion by NK cells to varying degrees. Among the 28 natural product candidates, we further confirmed and analyzed the potential of one molecule, andrographolide. It actually increased IFN-γ secretion by NK cells and enhanced NK cell-mediated killing of NSCLC cells. Our results demonstrated that this IFN-γ based high-throughput assay for screening of natural products for NK cell tumoricidal activity is a simple, economic and reliable method.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seong, Yeon-Jae; Hafis Clinic, Seoul; Sung, Pil Soo

Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced themore » production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.« less

Effect of kumquat (Fortunella crassifolia) pericarp on natural killer cell activity in vitro and in vivo.

PubMed

Nagahama, Kiyoko; Eto, Nozomu; Shimojo, Tomofumi; Kondoh, Tomomi; Nakahara, Keiko; Sakakibara, Yoichi; Fukui, Keiichi; Suiko, Masahito

2015-01-01

Natural killer (NK) cells play a key role in innate immune defense against infectious disease and cancer. A reduction of NK activity is likely to be associated with increased risk of these types of disease. In this study, we investigate the activation potential of kumquat pericarp acetone fraction (KP-AF) on NK cells. It is shown to significantly increase IFN-γ production and NK cytotoxic activity in human KHYG-1 NK cells. Moreover, oral administration of KP-AF significantly improves both suppressed plasma IFN-γ levels and NK cytotoxic activity per splenocyte in restraint-stressed mice. These results indicate that raw kumquat pericarp activates NK cells in vitro and in vivo. To identify the active constituents, we also examined IFN-γ production on KHYG-1 cells by the predicted active components. Only β-cryptoxanthin increased IFN-γ production, suggesting that NK cell activation effects of KP-AF may be caused by carotenoids such as β-cryptoxanthin.

Accumulation of Cytotoxic CD16+ NK Cells in Simian Immunodeficiency Virus-Infected Lymph Nodes Associated with In Situ Differentiation and Functional Anergy.

PubMed

Schafer, Jamie L; Li, Haiying; Evans, Tristan I; Estes, Jacob D; Reeves, R Keith

2015-07-01

Recent evidence suggests that even in treated infections, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication may continue in lymph nodes (LN), serving as a potential virus reservoir. Here we investigated the effects of lentivirus infection on natural killer (NK) cell frequencies, phenotypes, and functions in naive and acutely or chronically SIVmac239-infected rhesus macaques. Compared to that in naive animals, we observed a 3-fold-greater frequency of cytotoxic CD16(+) CD56(-) NK cells in LN of chronically infected macaques. However, NK cells did not appear to be trafficking to LN, as homing markers CD62L and CCR7 did not increase on circulating NK cells during infection. LN NK cells demonstrated enhanced cytotoxicity in acute infection, with 2-fold increases in perforin expression and 3-fold increases in CD107a expression following mitogen stimulation. Lysis of K562 cells by LN NK cells from acutely infected animals was greater than lysis by preinfection samples from the same animals. LN NK cells from chronically infected animals lysed K562 cells more efficiently than LN NK cells from uninfected animals, but importantly, surrogate markers of cytotoxicity in infected macaques were disproportionately greater than ex vivo killing. Furthermore, Tim-3, an indicator of activation and/or exhaustion, was upregulated 3-fold on LN NK cells in chronically infected animals. Collectively, these data suggest that LN NK cells are skewed toward a cytotoxic phenotype during SIV infection but may become dysfunctional and exhausted in chronic disease. The accumulation of CD16(+) CD56(-) NK cells in the SIV-infected lymph node without changes in NK homing to the LN could suggest that these cells are differentiating in situ. Surprisingly, this increase in frequency of the cytotoxic subset of NK cells is not accompanied by an increase of similar magnitude in the cytolytic function of LN lymphocytes. This functional modulation, together with the higher Tim-3 expression observed on LN NK cells isolated from chronically infected animals than on those from naive macaques, is indicative of an exhausted phenotype. This exhaustion could contribute to the robust replication of HIV and SIV in the LN during acute and chronic stages of infection, allowing the survival of infected cells and maintenance of a viral reservoir. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

NK cells influence both innate and adaptive immune responses after mucosal immunisation with antigen and mucosal adjuvant*

PubMed Central

Hall, Lindsay J; Clare, Simon; Dougan, Gordon

2012-01-01

NK cells were found to be recruited in a temporally controlled manner to the nasal-associated lymphoid tissue and the cervical lymph nodes of mice following intranasal immunisation with Ag85B-ESAT6 antigen from Mycobacterium tuberculosis mixed with Escherichia coli heat-labile toxin as adjuvant. These NK cells were activated and they secreted a diverse range of cytokines and other immunmodulators. Using antibody depletion targeting anti-asialo GM1, we found evidence for altered trafficking, impaired activation and cytokine secretion of dendritic cells, macrophages and neutrophils in immunised NK cell depleted mice compared to control animals. Analysis of antigen-specific immune responses revealed an attenuated antibody and cytokine response in immunised NK cell depleted animals. Systemic administration of rIL-6 but not rIFN-γ significantly restored immune responses in mice depleted of NK cells. In conclusion, cytokine production, particularly IL-6, via NK cells and NK cell activated immune populations, plays an important role in the establishment of local innate immune responses and the consequent development of adaptive immunity after mucosal immunisation. PMID:20220095

Altered phenotype and function of NK cells infiltrating human papillomavirus (HPV)-associated genital warts during HIV infection.

PubMed

Bere, Alfred; Tayib, Shahila; Kriek, Jean-Mari; Masson, Lindi; Jaumdally, Shameem Z; Barnabas, Shaun L; Carr, William H; Allan, Bruce; Williamson, Anna-Lise; Denny, Lynette; Passmore, Jo-Ann S

2014-02-01

HIV-infected individuals experience more persistent HPV infections and are less likely to resolve genital warts. This study compared phenotype and functions of NK and T cells from genital warts and blood from 67 women. We compared in vitro functional responses of NK and T cells by multiparametric flow cytometry. HIV+ women had significantly lower frequencies of CD4 T cells in warts (p = 0.001) and blood (p = 0.001). While the distribution of NK cell subsets was similar, HIV+ women tended to have lower frequencies of CD56(Dim) NK cells in both blood (p = 0.0001) and warts (p = 0.006) than HIV- women. Wart NK cells from HIV+ women expressed significantly lower CD107a and produced IFN-γ. HAART status was not associated with differences in NK cell functionality. We conclude that wart NK cells from HIV+ women have defects in their ability to degranulate and/or secrete IFN-γ, which may provide insights into why HIV+ women fail to spontaneously resolve genital warts. Copyright © 2013 Elsevier Inc. All rights reserved.

Natural Killer Cells in Obesity: Impaired Function and Increased Susceptibility to the Effects of Cigarette Smoke

PubMed Central

O'Shea, Donal; Cawood, Tom J.; O'Farrelly, Cliona; Lynch, Lydia

2010-01-01

Background Obese individuals who smoke have a 14 year reduction in life expectancy. Both obesity and smoking are independantly associated with increased risk of malignancy. Natural killer cells (NK) are critical mediators of anti-tumour immunity and are compromised in obese patients and smokers. We examined whether NK cell function was differentially affected by cigarette smoke in obese and lean subjects. Methodology and Principal Findings Clinical data and blood were collected from 40 severely obese subjects (BMI>40 kg/m2) and 20 lean healthy subjects. NK cell levels and function were assessed using flow cytometry and cytotoxicity assays. The effect of cigarette smoke on NK cell ability to kill K562 tumour cells was assessed in the presence or absence of the adipokines leptin and adiponectin. NK cell levels were significantly decreased in obese subjects compared to lean controls (7.6 vs 16.6%, p = 0.0008). NK function was also significantly compromised in obese patients (30% +/− 13% vs 42% +/−12%, p = 0.04). Cigarette smoke inhibited NK cell ability to kill tumour cell lines (p<0.0001). NK cells from obese subjects were even more susceptible to the inhibitory effects of smoke compared to lean subjects (33% vs 28%, p = 0.01). Cigarette smoke prevented NK cell activation, as well as perforin and interferon-gamma secretion upon tumour challenge. Adiponectin but not leptin partially reversed the effects of smoke on NK cell function in both obese (p = 0.002) and lean controls (p = 0.01). Conclusions/Significance Obese subjects have impaired NK cell activity that is more susceptible to the detrimental effects of cigarette smoke compared to lean subjects. This may play a role in the increase of cancer and infection seen in this population. Adiponectin is capable of restoring NK cell activity and may have therapeutic potential for immunity in obese subjects and smokers. PMID:20107494

In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer.

PubMed

Veluchamy, John P; Lopez-Lastra, Silvia; Spanholtz, Jan; Bohme, Fenna; Kok, Nina; Heideman, Daniëlle A M; Verheul, Henk M W; Di Santo, James P; de Gruijl, Tanja D; van der Vliet, Hans J

2017-01-01

Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) act by inhibiting EGFR downstream signaling and by eliciting a natural killer (NK) cell-mediated antitumor response. The IgG 1 mAb cetuximab has been used for treatment of RAS wt metastatic colorectal cancer (mCRC) patients, showing limited efficacy. In the present study, we address the potential of adoptive NK cell therapy to overcome these limitations investigating two allogeneic NK cell products, i.e., allogeneic activated peripheral blood NK cells (A-PBNK) and umbilical cord blood stem cell-derived NK cells (UCB-NK). While cetuximab monotherapy was not effective against EGFR - RAS wt , EGFR + RAS mut , and EGFR + BRAF mut cells, A-PBNK were able to initiate lysis of EGFR + colon cancer cells irrespective of RAS or BRAF status. Cytotoxic effects of A-PBNK (but not UCB-NK) were further potentiated significantly by coating EGFR + colon cancer cells with cetuximab. Of note, a significantly higher cytotoxicity was induced by UCB-NK in EGFR - RAS wt (42 ± 8 versus 67 ± 7%), EGFR + RAS mut (20 ± 2 versus 37 ± 6%), and EGFR + BRAF mut (23 ± 3 versus 43 ± 7%) colon cancer cells compared to A-PBNK and equaled the cytotoxic efficacy of the combination of A-PBNK and cetuximab. The antitumor efficacy of UCB-NK cells against cetuximab-resistant human EGFR + RAS mut colon cancer cells was further confirmed in an in vivo preclinical mouse model where UCB-NK showed enhanced antitumor cytotoxicity against colon cancer independent of EGFR and RAS status. As UCB-NK have been proven safe in a recently conducted phase I clinical trial in acute myeloid leukemia, a fast translation into clinical proof of concept for mCRC could be considered.

Two photon microscopy intravital study of DC-mediated anti-tumor response of NK cells

NASA Astrophysics Data System (ADS)

Caccia, Michele; Gorletta, Tatiana; Sironi, Laura; Zanoni, Ivan; Salvetti, Cristina; Collini, Maddalena; Granucci, Francesca; Chirico, Giuseppe

2010-02-01

Recent studies have demonstrated that dendritic cells (DCs) play a crucial role in the activation of Natural Killer cells (NKs) that are responsible for anti-tumor innate immune responses. The focus of this report is on the role of pathogen associated molecular pattern (PAMP) activated-DCs in inducing NK cell-mediated anti-tumor responses. Mice transplanted sub-cute (s.c.) with AK7 cells, a mesothelioma cell line sensitive to NK cell responses, are injected with fluorescent NK cells and DC activation is then induced by s.c. injection of Lipopolysaccharide (LPS). Using 4 dimensional tracking we follow the kinetic behavior of NK cells at the Draining Lymph-Node (DLN). As control, noninflammatory conditions are also evaluated. Our data suggest that NK cells are recruited to the DLN where they can interact with activated-DCs with a peculiar kinetic behavior: short lived interactions interleaved by rarer longer ones. We also found that the changes in the NK dynamic behavior in inflammatory conditions clearly affect relevant motility parameters such as the instantaneous and average velocity and the effective diffusion coefficient. This observation suggests that NK cells and activated-DCs might efficiently interact in the DLN, where cells could be activated. Therefore the interaction between activated-DCs and NK cells in DLN is not only a reality but it may be also crucial for the start of the immune response of the NKs.

Autocrine hemokinin-1 functions as endogenous adjuvant for IgE-mediated mast cell inflammatory responses

PubMed Central

Sumpter, Tina L.; Ho, Chin H.; Pleet, Anna R.; Tkacheva, Olga A.; Shufesky, William J.; Rojas-Canales, Darling M.; Morelli, Adrian E.; Larregina, Adriana T.

2014-01-01

Background Efficient development of atopic diseases requires interaction between allergen and adjuvant to initiate and amplify underlying inflammatory responses. Substance P (SP) and hemokinin-1 (HK-1) are neuropeptides that signal via the neurokinin-1 receptor (NK1R) to promote inflammation. Mast cells initiate the symptoms and tissue effects of atopic disorders, secreting TNF and IL-6 following FcεRI crosslinking by Ag-IgE complexes, (FcεRI-MCs). Additionally, MCs express the NK1R suggesting an adjuvant role of NK1R agonists for FcεRI-MC mediated pathologies, however in depth research addressing this relevant aspect of MC biology is lacking. Objective To investigate the effect of NK1R-signaling and the individual roles of SP and HK-1 as potential adjuvants for FcεRI-MC mediated allergic disorders. Methods Bone marrow (BM) MCs derived from C57BL/6-wild type (WT) or NK1R−/− mice were used to investigate the effects of NK1R signaling of FcεRI-activated MCs. BMMCs generated from Tac1−/− mice or following culture with Tac4 siRNA were used to address the adjuvancy of SP and HK-1. WT, NK1R−/− and c-KitW-sh/W-sh mice reconstituted with WT or NK1R−/− BMMCs were utilized to evaluate NK1R signaling on FcεRI-MC-mediated passive local and systemic anaphylaxis and airway inflammation. Results FcεRI-activated MCs up-regulated NK1R and HK-1 transcripts and protein synthesis, without modifying SP. In a positive signaling loop, HK-1 promoted TNF and IL6 secretion by MC degranulation and protein synthesis the later via the PI3K/Akt/NFκB pathways. In vivo, NK1R signaling was necessary for development of passive local and systemic anaphylaxis and chronic airway inflammation. Conclusions FcεRI-stimulation of MCs promotes autocrine secretion of HK-1 which signals via NK1R to provide adjuvancy for efficient development of FcεRI-MC-mediated disorders. PMID:25201259

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells.

PubMed

Davis, Zachary B; Cogswell, Andrew; Scott, Hamish; Mertsching, Amanda; Boucau, Julie; Wambua, Daniel; Le Gall, Sylvie; Planelles, Vicente; Campbell, Kerry S; Barker, Edward

2016-02-01

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94(+) NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94(+) NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL(+) CD56(dim) NK cells, in contrast to the efficient responses by CD56(bright) NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94(+) KIR2DL(-) NK cells may be uniquely beneficial.

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells

PubMed Central

Davis, Zachary B.; Cogswell, Andrew; Scott, Hamish; Mertsching, Amanda; Boucau, Julie; Wambua, Daniel; Le Gall, Sylvie; Planelles, Vicente; Campbell, Kerry S.; Barker, Edward

2016-01-01

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94+ NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94+ NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL+ CD56dim NK cells, in contrast to the efficient responses by CD56bright NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94+ KIR2DL- NK cells may be uniquely beneficial. PMID:26828202

Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the TH17 cytokine interleukin-22

PubMed Central

Hughes, Tiffany; Becknell, Brian; McClory, Susan; Briercheck, Edward; Freud, Aharon G.; Zhang, Xiaoli; Mao, Hsiaoyin; Nuovo, Gerard; Yu, Jianhua

2009-01-01

Considerable functional heterogeneity within human natural killer (NK) cells has been revealed through the characterization of distinct NK-cell subsets. Accordingly, a small subset of CD56+NKp44+NK cells, termed NK-22 cells, was recently described within secondary lymphoid tissue (SLT) as IL-22− when resting, with a minor fraction of this population becoming IL-22+ when activated. Here we discover that the vast majority of stage 3 immature NK (iNK) cells in SLT constitutively and selectively express IL-22, a TH17 cytokine important for mucosal immunity, whereas earlier and later stages of NK developmental intermediates do not express IL-22. These iNK cells have a surface phenotype of CD34−CD117+CD161+CD94−, largely lack expression of NKp44 and CD56, and do not produce IFN-γ or possess cytolytic activity. In summary, stage 3 iNK cells are highly enriched for IL-22 and IL-26 messenger RNA, and IL-22 protein production, but do not express IL-17A or IL-17F. PMID:19244159

IL-12-producing monocytes and HLA-E control HCMV-driven NKG2C+ NK cell expansion.

PubMed

Rölle, Alexander; Pollmann, Julia; Ewen, Eva-Maria; Le, Vu Thuy Khanh; Halenius, Anne; Hengel, Hartmut; Cerwenka, Adelheid

2014-12-01

Human cytomegalovirus (HCMV) infection is the most common cause of congenital viral infections and a major source of morbidity and mortality after organ transplantation. NK cells are pivotal effector cells in the innate defense against CMV. Recently, hallmarks of adaptive responses, such as memory-like features, have been recognized in NK cells. HCMV infection elicits the expansion of an NK cell subset carrying an activating receptor heterodimer, comprising CD94 and NKG2C (CD94/NKG2C), a response that resembles the clonal expansion of adaptive immune cells. Here, we determined that expansion of this NKG2C(+) subset and general NK cell recovery rely on signals derived from CD14(+) monocytes. In a coculture system, a subset of CD14(+) cells with inflammatory monocyte features produced IL-12 in response to HCMV-infected fibroblasts, and neutralization of IL-12 in this model substantially reduced CD25 upregulation and NKG2C(+) subset expansion. Finally, blockade of CD94/NKG2C on NK cells or silencing of the cognate ligand HLA-E in infected fibroblasts greatly impaired expansion of NKG2C(+) NK cells. Together, our results reveal that IL-12, CD14(+) cells, and the CD94/NKG2C/HLA-E axis are critical for the expansion of NKG2C(+) NK cells in response to HCMV infection. Moreover, strategies targeting the NKG2C(+) NK cell subset have the potential to be exploited in NK cell-based intervention strategies against viral infections and cancer.

Glycolipid presentation to natural killer T cells differs in an organ-dependent fashion

NASA Astrophysics Data System (ADS)

Schmieg, John; Yang, Guangli; Franck, Richard W.; van Rooijen, Nico; Tsuji, Moriya

2005-01-01

It has been shown that dendritic cells (DCs) are able to present glycolipids to natural killer (NK) T cells in vivo. However, the essential role of DCs, as well as the role of other cells in glycolipid presentation, is unknown. Here, we show that DCs are the crucial antigen-presenting cells (APCs) for splenic NK T cells, whereas Kupffer cells are the key APCs for hepatic NK T cells. Both cell types stimulate cytokine production by NK T cells within 2 h of glycolipid administration, but only DCs are involved in the systemic, downstream responses to glycolipid administration. More specifically, CD8+ DCs produce IL-12 in response to glycolipid presentation, which stimulates secondary IFN- production by NK cells in different organs. Different APCs participate in glycolipid presentation to NK T cells in vivo but differ in their involvement in the overall glycolipid response. dendritic cell | Kupffer cell

NK Cells and Their Role in Invasive Mold Infection.

PubMed

Schmidt, Stanislaw; Condorelli, Annalisa; Koltze, Antonia; Lehrnbecher, Thomas

2017-05-19

There is growing evidence that Natural Killer (NK) cells exhibit in vitro activity against both Aspergillus and non- Aspergillus molds. Cytotoxic molecules such as NK cell-derived perforin seem to play an important role in the antifungal activity. In addition, NK cells release a number of cytokines upon stimulation by fungi, which modulate both innate and adaptive host immune responses. Whereas the in vitro data of the antifungal activity of NK cells are supported by animal studies, clinical data are scarce to date.

Improving efficacy of cancer immunotherapy by genetic modification of natural killer cells.

PubMed

Burga, Rachel A; Nguyen, Tuongvan; Zulovich, Jane; Madonna, Sarah; Ylisastigui, Loyda; Fernandes, Rohan; Yvon, Eric

2016-11-01

Natural killer (NK) cells are members of the innate immune system that recognize target cells via activating and inhibitory signals received through cell receptors. Derived from the lymphoid lineage, NK cells are able to produce cytokines and exert a cytotoxic effect on viral infected and malignant cells. It is their unique ability to lyse target cells rapidly and without prior education that renders NK cells a promising effector cell for adoptive cell therapy. However, both viruses and tumors employ evasion strategies to avoid attack by NK cells, which represent biological challenges that need to be harnessed to fully exploit the cytolytic potential of NK cells. Using genetic modification, the function of NK cells can be enhanced to improve their homing, cytolytic activity, in vivo persistence and safety. Examples include gene modification to express chemokine, high-affinity Fc receptor and chimeric antigen receptors, suicide genes and the forced expression of cytokines such as interleukin (IL)-2 and IL-15. Preclinical studies have clearly demonstrated that such approaches are effective in improving NK-cell function, homing and safety. In this review, we summarize the recent advances in the genetic manipulations of NK cells and their application for cellular immunotherapeutic strategies. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Effects of first-line chemotherapy on natural killer cells in adult T-cell leukemia-lymphoma and peripheral T-cell lymphoma.

PubMed

Ogura, Michinori; Ishida, Takashi; Tsukasaki, Kunihiro; Takahashi, Takeshi; Utsunomiya, Atae

2016-07-01

Natural killer (NK) cells are well known to be the most important effector cells mediating antibody-dependent cellular cytotoxicity (ADCC) which is an important mechanism of action of antibody drugs. We evaluated the effects of chemotherapy on the cell number and activity of NK cells from patients who received the vincristine-cyclophosphamide-doxorubicin-prednisone (VCAP), doxorubicin-ranimustine-prednisone (AMP), and vindesine-etoposide-carboplatin-prednisone (VECP) (mLSG15) or mLSG15-like (-L) regimen, which is one of the standard of cares for newly diagnosed adult T-cell leukemia-lymphoma (ATL), or the cyclophosphamide-doxorubicin-vincristine-prednisone (CHOP) or CHOP-L regimen which is another standard of care for ATL and peripheral T-cell lymphoma (PTCL). The number of lymphocytes and NK cells, and NK cell activity, were assessed using flow cytometry and a (51)Cr release assay, respectively. A total of 26 patients with untreated ATL or PTCL were enrolled, and blood samples from 25 patients were evaluable. NK cell number in ATL decreased after mLSG15/-L treatment, and the degree of decrease in the NK cell number was more prominent just before VECP therapy (Day 15-17 of each cycle) than just before VCAP therapy (Day 1 of each cycle). The NK cell number in ATL after CHOP/-L treatment also decreased. Interestingly, the NK cell activity showed a tendency to increase after the treatment. NK cell number in PTCL did not decrease by CHOP/-L regimen, but the activity was slightly decreased after the treatment. These results indicate that the effects of chemotherapeutic agents on NK cells vary according to the disease type and intensity of chemotherapy.

Differences in the length of the carboxyl terminus mediate functional properties of neurokinin-1 receptor

PubMed Central

Lai, Jian-Ping; Lai, Saien; Tuluc, Florin; Tansky, Morris F.; Kilpatrick, Laurie E.; Leeman, Susan E.; Douglas, Steven D.

2008-01-01

The neurokinin-1 receptor (NK1R) has two naturally occurring forms that differ in the length of the carboxyl terminus: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. We examined whether there are differential signaling properties attributable to the carboxyl terminus of this receptor by using stably transfected human embryonic kidney (HEK293) cell lines that express either full-length or truncated NK1R. Substance P (SP) specifically triggered intracellular calcium increase in HEK293 cells expressing full-length NK1R but had no effect in the cells expressing the truncated NK1R. In addition, in cells expressing full-length NK1R, SP activated NF-κB and IL-8 mRNA expression, but in cells expressing the truncated NK1R, SP did not activate NF-κB, and it decreased IL-8 mRNA expression. In cells expressing full-length NK1R, SP stimulated phosphorylation of PKCδ but inhibited phosphorylation of PKCδ in cells expressing truncated NK1R. There are also differences in the timing of SP-induced ERK activation in cells expressing the two different forms of the receptor. Full-length NK1R activation of ERK was rapid (peak within 1–2 min), whereas truncated NK1R-mediated activation was slower (peak at 20–30 min). Thus, the carboxyl terminus of NK1R is the structural basis for differences in the functional properties of the full-length and truncated NK1R. These differences may provide important information toward the design of new NK1R receptor antagonists. PMID:18713853

Blastic natural killer cell leukaemia in a dog--a case report.

PubMed

Bonkobara, Makoto; Saito, Taro; Yamashita, Masahiro; Tamura, Kyoichi; Yagihara, Hiroko; Isotani, Mayu; Sato, Takashi; Washizu, Tsukimi

2007-11-01

A case of canine non-T, non-B lymphoid leukaemia was determined to be of natural killer (NK) cell lineage by detecting specific expression of canine CD56 mRNA by reverse transcriptase polymerase chain reaction analysis. Although NK cells are usually considered to be morphologically large granular lymphocytes, the malignant NK cells in this case were agranular and blast-like, resembling human blastic NK cell leukaemia. The prognosis of human NK cell leukaemia is usually poor. In this case, the dog died 10 days after initial presentation, despite chemotherapy.

Mechanisms of diminished natural killer cell activity in pregnant women and neonates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baley, J.E.; Schacter, B.Z.

1985-05-01

Because alterations in natural killer (NK) activity in the perinatal period may be important in the maintenance of a healthy pregnancy, the mechanisms by which these alterations are mediated in neonates and in pregnant and postpartum women was examined. NK activity, as measured in a 4-hr /sup 51/Cr-release assay and compared with adult controls, is significantly diminished in all three trimesters of pregnancy and in immediately postpartum women. In postpartum women, NK activity appears to be higher than in pregnant women, although this does not reach statistical significance. Pregnant and postpartum women have normal numbers of large granular lymphocytes andmore » normal target cell binding in an agarose single cell assay but decreased lysis of the bound target cells. NK activity of mononuclear cells from postpartum women, in addition, demonstrate a shift in distribution to higher levels of resistance to gamma-irradiation. Further, sera from postpartum women cause a similar shift to increased radioresistance in mononuclear cells from adult controls. Because radioresistance is a property of interleukin 2-stimulated NK, the shift to radioresistance may represent lymphokine-mediated stimulation occurring during parturition. In contrast, cord blood cells have a more profound decrease in NK activity as determined by /sup 51/Cr-release assay and decreases in both binding and lysis of bound target cells in the single cell assay. The resistance of NK activity in cord cells to gamma-irradiation is also increased, as seen in postpartum women. Cord blood serum, however, did not alter radioresistance or inhibit NK activity. The results suggest that the observed diminished NK activity in pregnant women and neonates arise by different mechanisms: an absence of mature NK cells in the neonate and an alteration of the NK cell in pregnancy leading to decreased killing.« less

Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming.

PubMed

Capuano, Cristina; Battella, Simone; Pighi, Chiara; Franchitti, Lavinia; Turriziani, Ombretta; Morrone, Stefania; Santoni, Angela; Galandrini, Ricciarda; Palmieri, Gabriella

2018-01-01

Natural killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C + FcεRIγ - long-lived "memory" NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV-seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterized yet. Here, we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells and we show the impact of donor' HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansions. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy.

2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy

PubMed Central

Ostrowski, S R; Ullum, H; Pedersen, B K; Gerstoft, J; Katzenstein, T L

2005-01-01

Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected by highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with ≤ 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4 expression on CD3− CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected patients (P < 0·001) but increased to a normal level during the 24 months’ follow-up. The concentration of CD3+ CD8+ 2B4+ cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during follow-up (both P < 0·001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0·05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B4+ NK cells normalizes during long-term HAART in HIV-1 infected patients. The finding that proviral-DNA and sTNFrII were associated negatively with the concentration of 2B4+ NK cells suggests that immune activation in HIV-1 infected patients receiving HAART influences the target cell recognition by NK cells. PMID:16045743

2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy.

PubMed

Ostrowski, S R; Ullum, H; Pedersen, B K; Gerstoft, J; Katzenstein, T L

2005-09-01

Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected by highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with < or = 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4 expression on CD3- CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected patients (P < 0.001) but increased to a normal level during the 24 months' follow-up. The concentration of CD3+ CD8+ 2B4+ cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during follow-up (both P < 0.001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0.05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B4+ NK cells normalizes during long-term HAART in HIV-1 infected patients. The finding that proviral-DNA and sTNFrII were associated negatively with the concentration of 2B4+ NK cells suggests that immune activation in HIV-1 infected patients receiving HAART influences the target cell recognition by NK cells.

Effects of ADAM10 and ADAM17 Inhibitors on Natural Killer Cell Expansion and Antibody-dependent Cellular Cytotoxicity Against Breast Cancer Cells In Vitro.

PubMed

Pham, Dang-Huan; Kim, Ju-Sun; Kim, Sang-Ki; Shin, Dong-Jun; Uong, Nguyen-Thanh-Tung; Hyun, Hoon; Yoon, Mee Sun; Kang, Sin Jae; Ryu, Young Jae; Cho, Jin Seong; Yoon, Jung Han; Lee, Ji Shin; Cho, Duck; Lee, Soo-Hyeon; Park, Min Ho

2017-10-01

The inhibition of a disintegrin and metalloproteinase (ADAM) has the potential to become a novel approach for natural killer (NK) cell-based cancer immunotherapy. Thus, the aim of this study was to investigate the influence of ADAM10 and ADAM17 inhibitors on expanded NK cell to enhance antibody-dependent cellular cytotoxicity (ADCC) in breast cancer cell lines. NK cells were expanded in medium supplemented with an ADAM10 or ADAM17 inhibitor to prevent the shedding of soluble CD16/FcγRIII. The expression level of CD16 and production of interferon-gamma (IFN-γ) was detected by flow cytometry using specific antibodies. ADCC activity of expanded NK cells was estimated in trastuzumab treated breast cancer cell lines such as MCF-7, MDA-MB-231, SKBR3, and BT-474 cells. The ADAM17 inhibitor increased the purity of expanded NK cells to 90% after 14 days at 5 and 10 μM in vitro (p=0.043). However, the expansion rate of NK cells was decreased at 10 μM of the ADAM 17 inhibitor (p=0.043). Inhibition of ADAM10 suppressed the expansion of NK cells, although the NK purity was increased at 1 μM of the inhibitor. The expression of CD16 was significantly increased at 1 and 5 μM of the ADAM17 inhibitor (p=0.046, 0.028, respectively) during the culturing period. Inhibition of ADAM10 reduced the expression of CD16 on NK cells. The cytotoxic activity of the ADAM17 inhibitor treated NK cells against MCF-7 (p=0.039) and BT-474 (p=0.027) cells was significantly elevated. The ADCC activity of NK cells treated with 5 μM of ADAM17 inhibitor was significantly increased against SKBR-3 and BT-474 (p=0.027). Inhibition of ADAM17 increased the production of IFN-γ in expanded NK cells. The inhibition of ADAM17 enhanced the purity of expanded NK cells and the ADCC activity of these cells against trastuzumab treated breast cancer cell lines. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E.

PubMed Central

Valés-Gómez, M; Reyburn, H T; Erskine, R A; López-Botet, M; Strominger, J L

1999-01-01

The lytic function of human natural killer (NK) cells is markedly influenced by recognition of class I major histocompatibility complex (MHC) molecules, a process mediated by several types of activating and inhibitory receptors expressed on the NK cell. One of the most important of these mechanisms of regulation is the recognition of the non-classical class I MHC molecule HLA-E, in complex with nonamer peptides derived from the signal sequences of certain class I MHC molecules, by heterodimers of the C-type lectin-like proteins CD94 and NKG2. Using soluble, recombinant HLA-E molecules assembled with peptides derived from different leader sequences and soluble CD94/NKG2-A and CD94/NKG2-C proteins, the binding of these receptor-ligand pairs has been analysed. We show first that these interactions have very fast association and dissociation rate constants, secondly, that the inhibitory CD94/NKG2-A receptor has a higher binding affinity for HLA-E than the activating CD94/NKG2-C receptor and, finally, that recognition of HLA-E by both CD94/NKG2-A and CD94/NKG2-C is peptide dependent. There appears to be a strong, direct correlation between the binding affinity of the peptide-HLA-E complexes for the CD94/NKG2 receptors and the triggering of a response by the NK cell. These data may help to understand the balance of signals that control cytotoxicity by NK cells. PMID:10428963

Impaired NK Cell Responses to Pertussis and H1N1 Influenza Vaccine Antigens in Human Cytomegalovirus-Infected Individuals

PubMed Central

Nielsen, Carolyn M.; White, Matthew J.; Bottomley, Christian; Lusa, Chiara; Rodríguez-Galán, Ana; Turner, Scarlett E. G.; Goodier, Martin R.

2015-01-01

NK cells contribute to postvaccination immune responses after activation by IL-2 from Ag-specific memory T cells or by cross-linking of the low-affinity IgG receptor, CD16, by Ag–Ab immune complexes. Sensitivity of NK cells to these signals from the adaptive immune system is heterogeneous and influenced by their stage of differentiation. CD56dimCD57+ NK cells are less responsive to IL-2 and produce less IFN-γ in response to T cell–mediated activation than do CD56bright or CD56dimCD57− NK cells. Conversely, NK cell cytotoxicity, as measured by degranulation, is maintained across the CD56dim subsets. Human CMV (HCMV), a highly prevalent herpes virus causing lifelong, usually latent, infections, drives the expansion of the CD56dimCD57+NKG2C+ NK cell population, skewing the NK cell repertoire in favor of cytotoxic responses at the expense of cytokine-driven responses. We hypothesized, therefore, that HCMV seropositivity would be associated with altered NK cell responses to vaccine Ags. In a cross-sectional study of 152 U.K. adults, with HCMV seroprevalence rate of 36%, we find that HCMV seropositivity is associated with lower NK cell IFN-γ production and degranulation after in vitro restimulation with pertussis or H1N1 influenza vaccine Ags. Higher expression of CD57/NKG2C and lower expression of IL-18Rα on NK cells from HCMV seropositive subjects do not fully explain these impaired responses, which are likely the result of multiple receptor–ligand interactions. This study demonstrates for the first time, to our knowledge, that HCMV serostatus influences NK cell contributions to adaptive immunity and raises important questions regarding the impact of HCMV infection on vaccine efficacy. PMID:25855356

Distinct Upstream Role of Type I IFN Signaling in Hematopoietic Stem Cell-Derived and Epithelial Resident Cells for Concerted Recruitment of Ly-6Chi Monocytes and NK Cells via CCL2-CCL3 Cascade

PubMed Central

Choi, Jin Young; Kim, Seong Bum; Eo, Seong Kug

2015-01-01

Type I interferon (IFN-I)-dependent orchestrated mobilization of innate cells in inflamed tissues is believed to play a critical role in controlling replication and CNS-invasion of herpes simplex virus (HSV). However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown. Here, we found that IFN-I signaling promoted the differentiation of CCL2-producing Ly-6Chi monocytes and IFN-γ/granzyme B-producing NK cells, whereas deficiency of IFN-I signaling induced Ly-6Clo monocytes producing CXCL1 and CXCL2. More interestingly, recruitment of Ly-6Chi monocytes preceded that of NK cells with the levels peaked at 24 h post-infection in IFN-I–dependent manner, which was kinetically associated with the CCL2-CCL3 cascade response. Early Ly-6Chi monocyte recruitment was governed by CCL2 produced from hematopoietic stem cell (HSC)-derived leukocytes, whereas NK cell recruitment predominantly depended on CC chemokines produced by resident epithelial cells. Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6Ghi neutrophil recruitment to ameliorate immunopathology. Finally, tissue resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ dendritic cells appeared to produce initial CCL2 for migration-based self-amplification of early infiltrated Ly-6Chi monocytes upon stimulation by IFN-I produced from infected epithelial cells. Ultimately, these results decipher a detailed IFN-I–dependent pathway that establishes orchestrated mobilization of Ly-6Chi monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells. Therefore, this cascade response of resident–to-hematopoietic–to-resident cells that drives cytokine–to-chemokine–to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues. PMID:26618488

IL-12–producing monocytes and HLA-E control HCMV-driven NKG2C+ NK cell expansion

PubMed Central

Rölle, Alexander; Pollmann, Julia; Ewen, Eva-Maria; Le, Vu Thuy Khanh; Halenius, Anne; Hengel, Hartmut; Cerwenka, Adelheid

2014-01-01

Human cytomegalovirus (HCMV) infection is the most common cause of congenital viral infections and a major source of morbidity and mortality after organ transplantation. NK cells are pivotal effector cells in the innate defense against CMV. Recently, hallmarks of adaptive responses, such as memory-like features, have been recognized in NK cells. HCMV infection elicits the expansion of an NK cell subset carrying an activating receptor heterodimer, comprising CD94 and NKG2C (CD94/NKG2C), a response that resembles the clonal expansion of adaptive immune cells. Here, we determined that expansion of this NKG2C+ subset and general NK cell recovery rely on signals derived from CD14+ monocytes. In a coculture system, a subset of CD14+ cells with inflammatory monocyte features produced IL-12 in response to HCMV-infected fibroblasts, and neutralization of IL-12 in this model substantially reduced CD25 upregulation and NKG2C+ subset expansion. Finally, blockade of CD94/NKG2C on NK cells or silencing of the cognate ligand HLA-E in infected fibroblasts greatly impaired expansion of NKG2C+ NK cells. Together, our results reveal that IL-12, CD14+ cells, and the CD94/NKG2C/HLA-E axis are critical for the expansion of NKG2C+ NK cells in response to HCMV infection. Moreover, strategies targeting the NKG2C+ NK cell subset have the potential to be exploited in NK cell–based intervention strategies against viral infections and cancer. PMID:25384219