Characterization of Human NK Cell-Derived Exosomes: Role of DNAM1 Receptor in Exosome-Mediated Cytotoxicity against Tumor

Despite the pivotal role of natural killer (NK) cells in defenses against tumors, their exploitation in cancer treatment is still limited due to their reduced ability to reaching tumor sites and the inhibitory effects of tumor microenvironment (TME) on their function. In this study, we have characterized the exosomes from IL2- or IL15-cultured human NK cells. Both cytokines induced comparable amounts of exosomes with similar cargo composition. Analysis of molecules contained within or exposed at the exosome surface, allowed the identification of molecules playing important roles in the NK cell function including IFN-γ, Lymphocyte Function-Associated Antigen (LFA-1), DNAX Accessory Molecule-1 (DNAM1) and Programmed Cell Death Protein (PD-1). Importantly, we show that DNAM1 is involved in exosome-mediated cytotoxicity as revealed by experiments using blocking antibodies to DNAM1 or DNAM1 ligands. In addition, antibody-mediated inhibition of exosome cytotoxicity results in a delay in target cell apoptosis. We also provide evidence that NK-exosomes may exert their cytolytic activity after short time interval and even at low concentrations. Regarding their possible use in immunotherapy, NK exosomes, detectable in peripheral blood, can diffuse into tissues and exert their cytolytic effect at tumor sites. This property offers a clue to integrate cancer treatments with NK exosomes.

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Advanced Materials and Devices for the Regulation and Study of NK Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20030646 ◽

2019 ◽

Vol 20 (3) ◽

pp. 646 ◽

Cited By ~ 2

Author(s):

Guillaume Le Saux ◽

Mark Schvartzman

Keyword(s):

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Advanced Materials ◽

Immune Protection ◽

Adaptive Responses ◽

Environmental Signals ◽

Recent Advances ◽

Innate Lymphocytes

Natural Killer (NK) cells are innate lymphocytes that contribute to immune protection by cytosis, cytokine secretion, and regulation of adaptive responses of T cells. NK cells distinguish between healthy and ill cells, and generate a cytotoxic response, being cumulatively regulated by environmental signals delivered through their diverse receptors. Recent advances in biomaterials and device engineering paved the way to numerous artificial microenvironments for cells, which produce synthetic signals identical or similar to those provided by the physiological environment. In this paper, we review recent advances in materials and devices for artificial signaling, which have been applied to regulate NK cells, and systematically study the role of these signals in NK cell function.

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Costimulatory Effects of Interleukin-18 (IL-18) on Human Natural Killer (NK) Cells Activated through Fc Receptors for Immunoglobulin (IgG)

Blood ◽

10.1182/blood.v116.21.2780.2780 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2780-2780

Author(s):

Shivani Srivastava ◽

Hailin Feng ◽

Menggang Yu ◽

David Pelloso ◽

Michael Robertson

Keyword(s):

Monoclonal Antibodies ◽

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Conflicts Of Interest ◽

Fc Receptors ◽

Cytolytic Activity ◽

Mitogen Activated Protein Kinase ◽

Target Cells

Abstract Abstract 2780 NK cells play an important role in innate and adaptive immune responses. Most human NK cells express CD16, an Fc receptor for IgG that mediates lysis of antibody-coated target cells and costimulates interferon (IFN)-g production in response to cytokines. IL-18 is an immunostimulatory cytokine with antitumor activity in preclinical animal models. The effects of IL-18 on human NK cell function were examined. Here we show that NK cells stimulated with immobilized IgG in vitro secreted IFN-g; such IFN-g production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-g production by NK cells stimulated with immobilized IgG or CD16 antibodies (Figure 1). NK cell IFN-g production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk, extracellular signal-related kinases (ERK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3-K). Stimulation with IL-18 or immobilized IgG could augment IL-12-induced IFN-g production by STAT4-deficient lymphocytes obtained from lymphoma patients after autologous stem cell transplantation (Figure 2). IL-18 also augmented the in vitro lysis of rituximab-coated Raji cells by human NK cells (Figure 3). These observations that IL-18 can co stimulate IFN-g production and cytolytic activity of NK cells activated through Fc receptors makes it an attractive cytokine to combine with monoclonal antibodies for treatment of cancer. Disclosure: No relevant conflicts of interest to declare.

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The Important Role of Dendritic Cell (DC) in iNKT-Mediated Modulation of NK Cell Function in Chlamydia pneumoniae Lung Infection

Mediators of Inflammation ◽

10.1155/2019/4742634 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 2

Author(s):

Lei Zhao ◽

Xiaoling Gao ◽

Hong Bai ◽

Antony George Joyee ◽

Shuhe Wang ◽

...

Keyword(s):

Nk Cells ◽

Adoptive Transfer ◽

Chlamydia Pneumoniae ◽

Cell Function ◽

Nk Cell ◽

Lung Infection ◽

Inkt Cell ◽

Cellular Interactions ◽

Activation Markers

Chlamydia pneumoniae(Cpn) infection causes multiple acute and chronic human diseases. The role of DCs in host defense against Cpn infection has been well documented. The same is true for invariant natural killer T (iNKT) cells and NK cells, but the interaction among cells is largely unknown. In this study, we investigated the influence and mechanism of iNKT cell on the differentiation and function of NK cell inCpnlung infection and the role played by DCs in this process. We found that expansion of IFN-γ-producing NK cells quickly happened after the infection, but this response was altered in iNKT knockout (KO) mice. The expression of activation markers and the production of IFN-γby different NK subsets were significantly lower in KO mice than wild-type (WT) mice. Using in vitro DC-NK coculture and in vivo adoptive transfer approaches, we further examined the role of DCs in iNKT-mediated modulation of NK cell function. We found that NK cells expressed lower levels of activation markers and produced less IFN-γwhen they were cocultured with DCs from KO mice than WT mice. More importantly, we found that the adoptive transfer of DCs from the KO mice induced less NK cell activation and IFN-γproduction. The results provided evidence on the modulating effect of iNKT cell on NK cell function, particularly the critical role of DCs in this modulation process. The finding suggests the complexity of cellular interactions inCpnlung infection, which should be considered in designing preventive and therapeutic approaches for diseases and infections.

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Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells.

Journal of Experimental Medicine ◽

10.1084/jem.169.4.1373 ◽

1989 ◽

Vol 169 (4) ◽

pp. 1373-1389 ◽

Cited By ~ 245

Author(s):

W H Chambers ◽

N L Vujanovic ◽

A B DeLeo ◽

M W Olszowy ◽

R B Herberman ◽

...

Keyword(s):

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Cytolytic Activity ◽

Lak Cells ◽

Target Cells ◽

Lymphokine Activated Killer Cells ◽

Killer Cells ◽

And Function ◽

Lak Activity

To study the cellular structures involved in NK and lymphokine-activated killer (LAK) cell function, we have produced a panel of mAbs that modulate the cytolytic function of a population of cells with LAK activity that derive from large granular lymphocyte (LGL)/NK cells (adherent LAK [A-LAK] cells). In this report, we describe an mAb (3.2.3; IgG1k) that recognizes a triggering structure that is expressed on rat LGL/NK cells and A-LAK cells. This epitope is also expressed on polymorphonuclear leukocytes (PMN). The expression of the epitope identified by mAb 3.2.3 increased progressively on A-LAK cells after culture in the presence of rIL-2. mAb 3.2.3 enhanced the cytolytic activity of NK and A-LAK cells against FcR+ target cells, but not FcR- target cells. However, this effect was not induced by F(ab')2 fragments of 3.2.3. This antibody also induced the release of N-alpha-benzyloxycarbonyl-L-lysine thiobenzy esteresterase by A-LAK cells. These data suggest that the epitope identified by mAb 3.2.3 is on a triggering structure expressed on rat NK cells and A-LAK cells. The expression of the epitope recognized by mAb 3.2.3 on LGL/NK cells and PMN suggests that this structure may be analogous to that identified by the anti-CD16 (-FcR) mAbs. However, the molecule immunoprecipitated by mAb 3.2.3 was a 60-kD dimer composed of two 30-kD chains. These data suggest that mAb 3.2.3 recognizes a unique triggering structure. As mAb 3.2.3 is the first antibody recognizing a determinant with functional significance, selectively expressed on both rat NK cells and A-LAK cells, it will be a useful tool for the study of NK cell ontogeny and function, and the development of cells with LAK activity from the NK cell compartment.

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Ex Vivo IL-2 Expansion of CB-NK Cells Promotes Synergistic LFA-1 and CD2 Engagement at the NK Cell Lytic Immune Synapse; Implications for Adoptive CB-NK Cell Therapy in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.3029.3029 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3029-3029

Author(s):

Dongxia Xing ◽

Alan G. Ramsay ◽

William Decker ◽

Sufang Li ◽

Simon Robinson ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Synapse Formation ◽

Immunological Synapse ◽

Nk Cell ◽

Ex Vivo ◽

Immune Therapy ◽

Cytolytic Activity ◽

Lymphocyte Function ◽

Immune Synapse

Abstract Abstract 3029 Poster Board II-1005 Donor peripheral blood (PB) natural killer (NK) cell have shown clinical promise in cancer immunotherapy. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. Umbilical cord blood (CB) represents an important alternative source of NK cells for adoptive immune therapy. We first demonstrated that cord blood (CB) derived NK cells have poor cytolytic activity and deficiency in the formation of the F-actin immunological synapse with HLA class I deficient target K562 cells and primary AML blasts compared to PB-NK cells. In this study, we explored the cellular mechanism of these dysfunctions. We hypothesized that adhesion and signaling molecules may be defective in unmanipulated CB NK cells. Activating receptor Both CD2 and the integrin lymphocyte function-associated antigen (LFA-1) play important roles in both T lymphocyte and NK cell immune synapse formation and their trafficking to the immune synapse regulates both T and NK cell function. We now show that unmanipulated CB NK cells exhibit reduced LFA-1 mediated adhesion to mobilized ICAM-1 compared to IL-2 expanded CB NK cells (CB NK 29.7+/- 3.2 %, vs expanded CB NK 78.5+/- 6.1%, n=6). Moreover, unmanipulated CB-NK cells demonstrated reduced surface expression of CD2, and high affintyLFA-1 detected by the specific antibody (MHM24). There was decreased recruitment of CD2 and LFA-1 to the NK cell immune synapse site as quantified by confocal microscope analysis (RRI CD2 CB NK 2.02 vs PB NK 4.98, n=3). Furthermore, defective LFA-1 trafficking lead to a decrease in downstream cytotoxic granules that traffic to the immunological synapse as demonstrated by decreased perforin trafficking to the CB-NK synapse site (> 60% reduction).We next wanted to confirm that CD2 or LFA-1 play a role in restoring the immune synapseformation for IL-2 expanded CB NK cells. We incubated expanded CB NK cells with blocking antibodies specific for LFA-1 or CD2 prior to conjugation to the K562 target cells. After CD2 or LFA-1 blocking there was decreased synapse formation, with a resultant decrease in cytotoxic function. When monoclonal antibodies against both CD2 and LFA-1 were used there was significant blockade of the formation of the immune synapse, and a marked reduction of CB NK cell cytolytic activity (Mean specific lysis of K562 targets at E:T ratio 20:1 was 81% IgG control vs 22% with anti-CD2; and 29% with anti-LFA-1, n=6, P<0.001). This data shows that CD2 and LFA-1 are defective in unmanipulated CB NK cells resulting in impaired immune synapse formation. In contrast, ex vivo IL-2 expansion of CB-NK cells enhanced lytic synapse formation with the synergistic repair of CD2 and LFA-1 localization and activity. We believe our results provide important mechanistic insights for the potential use of IL-2 expanded CB-derived NK cells for adoptive immune therapy in leukemia. Disclosures No relevant conflicts of interest to declare.

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Response of human natural killer (NK) cells to NK cell stimulatory factor (NKSF): cytolytic activity and proliferation of NK cells are differentially regulated by NKSF.

Journal of Experimental Medicine ◽

10.1084/jem.175.3.779 ◽

1992 ◽

Vol 175 (3) ◽

pp. 779-788 ◽

Cited By ~ 231

Author(s):

M J Robertson ◽

R J Soiffer ◽

S F Wolf ◽

T J Manley ◽

C Donahue ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Function ◽

Nk Cell ◽

Human Peripheral Blood ◽

Killer Cell ◽

Interleukin 2 ◽

Cytolytic Activity ◽

Stimulatory Factor

Natural killer cell stimulatory factor (NKSF) is a 70-kD heterodimeric cytokine that was initially isolated from conditioned medium of human B lymphoblastoid cell lines. The effects of recombinant NKSF on the function of human peripheral blood NK cells were examined. NKSF directly augmented the cytolytic activity of freshly isolated NK cells. Both CD56dim and CD56bright NK cells demonstrated enhanced cytotoxicity after brief exposure to NKSF. In contrast, highly purified T lymphocytes did not exhibit major histocompatibility complex-unrestricted cytotoxicity after short-term culture with NKSF. Like interleukin 2 (IL-2), NKSF augmented the lysis of NK-sensitive, NK-resistant, and antibody-coated targets. Both NKSF and IL-2 induced marked upregulation of several NK cell adhesion molecules known to participate in cytolysis, including CD2, CD11a, and CD54. However, NKSF activates NK cells through a pathway distinct from that of IL-2, since the presence of anti-IL-2 receptor (anti-IL-2R) antibodies or IL-4 did not inhibit the effects of NKSF. NKSF by itself induced very little proliferation of resting NK cells. NK cells preactivated in vitro with IL-2 demonstrated enhanced proliferation to NKSF, but the degree of proliferation was always inferior to that induced by IL-2 alone. Moreover, NKSF strongly inhibited IL-2-induced proliferation of either resting or preactivated NK cells. This inhibition was not the result of decreased IL-2R expression, because NKSF-activated NK cells expressed higher levels of both IL-2Rs p75 and p55. Furthermore, NKSF did not inhibit the proliferation of mitogen-activated T cells, indicating a selective effect on NK cell proliferation. Human NK cells expanded in vivo by prolonged continuous infusions of IL-2 remained fully responsive to NKSF. Picomolar concentrations of NKSF were as effective as nanomolar concentrations of IL-2 in augmenting the cytolytic activity of NK cells expanded in vivo by IL-2. NKSF may play an important role in the regulation of human NK cell function, and its possible use as a therapeutic cytokine deserves further investigation.

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Key Role of the CD56lowCD16low Natural Killer Cell Subset in the Recognition and Killing of Multiple Myeloma Cells

Cancers ◽

10.3390/cancers10120473 ◽

2018 ◽

Vol 10 (12) ◽

pp. 473 ◽

Cited By ~ 12

Author(s):

Elisabetta Vulpis ◽

Helena Stabile ◽

Alessandra Soriani ◽

Cinzia Fionda ◽

Maria Petrucci ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Cell Subset ◽

Cytolytic Activity ◽

Hematopoietic Stem ◽

Natural Killer Cell Subset

Natural Killer (NK) cells play a pivotal role in the immunosurveillance of Multiple Myeloma (MM), but it is still undefined whether the NK cell functional properties underlying their protective activity against MM are confined to distinct NK cell populations. Interestingly, herein we report that the CD56lowCD16low NK cell subset displayed higher cytolytic activity compared to the other NK cell subsets (i.e., CD56highCD16+/−, CD56lowCD16high) against MM cells and its activity was impaired in MM patients. Decreased DNAM-1 expression levels were observed on the CD56lowCD16low NK cells during MM progression. Evaluating NK cell subset frequency after autologous hematopoietic stem cell transplantation, we found that CD56lowCD16low NK cells recovered earlier after transplantation. Overall, our data denote a key role of CD56lowCD16low subpopulation in the killing of MM cells and suggest that the reconstitution of CD56lowCD16low subpopulation after HSCT could be a useful approach of adoptive immunotherapy in the treatment of relapsed/refractory MM patients.

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The role of cytokine in regulation of the natural killer cell activity

Srpski arhiv za celokupno lekarstvo ◽

10.2298/sarh0808423j ◽

2008 ◽

Vol 136 (7-8) ◽

pp. 423-429 ◽

Cited By ~ 5

Author(s):

Vladimir Jurisic ◽

Sladjana Stojacic-Djenic ◽

Natasa Colovic ◽

Gordana Konjevic

Keyword(s):

Immune System ◽

Nk Cells ◽

Natural Killer ◽

Cell Function ◽

Nk Cell ◽

Natural Killer Cell Activity ◽

Killer Cell ◽

Cell Activity ◽

Target Cells

Natural killer (NK) cells are characterized by a CD3-CD16+ CD56+ immunophenotype and have a central role in the innate immune system. They are defined by their capacity to kill certain tumor-target cells or virus infected cells without prior sensitization or MHC-restriction. The activity of the NK cells is determined by the balance between activation and inhibitory receptor molecules expressed on the surface of NK cells. However, several cytokines and chemokines can significantly modulate their activity, inducing increase of NK cell activity. Immunomodulation mediated by NK cells is very important mechanism in tumor immunity, as well as in other immunodepressions of the immune system. In this study, we summarize the role of several cytokines, including IFN, IL-1, IL-2, IL-4, IL-7, IL-12 and IL-17, on NK cell function. The NK cells, after activation, depending on cytokine environment, can differentiate into NK1 cells that produce Th1 cytokine type (IFN-?, IL-2, IL-12) or NK2 cells that produce Th2 type cytokines, enhance exocytosis and release of previously formed molecules from NK cells (granzyme, perforin). We also describe that the release of cytokines and mediators show local or distance effects, or induce apoptosis (mostly by secreted TNF-?) after binding appropriated killer cell receptors from TNF receptor superfamily.

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The Role of Natural Killer Cells as a Platform for Immunotherapy in Pediatric Cancers

Current Oncology Reports ◽

10.1007/s11912-019-0837-8 ◽

2019 ◽

Vol 21 (10) ◽

Cited By ~ 3

Author(s):

Miriam Santiago Kimpo ◽

Bernice Oh ◽

Shawn Lee

Keyword(s):

Natural Killer Cells ◽

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Cell Function ◽

Nk Cell ◽

Childhood Cancers ◽

Killer Cells ◽

Cell Immunotherapy

Abstract Purpose of Review We aim to review the most recent findings in the use of NK cells in childhood cancers. Recent Findings Natural killer cells are cytotoxic to tumor cells. In pediatric leukemias, adoptive transfer of NK cells can bridge children not in remission to transplant. Interleukins (IL2, IL15) can enhance NK cell function. NK cell-CAR therapy has advantages of shorter life span that lessens chronic toxicities, lower risk of graft versus host disease when using allogeneic cells, ability of NK cells to recognize tumor cells that have downregulated MHC to escape T cells, and possibly less likelihood of cytokine storm. Cytotoxicity to solid tumors (rhabdomyosarcoma, Ewing’s sarcoma, neuroblastoma) is seen with graft versus tumor effect in transplant and in combination with antibodies. Challenges lie in the microenvironment which is suppressive for NK cells. Summary NK cell immunotherapy in childhood cancers is promising and recent works aim to overcome challenges.

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NK cells clear α-synuclein and the depletion of NK cells exacerbates synuclein pathology in a mouse model of α-synucleinopathy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909110117 ◽

2020 ◽

Vol 117 (3) ◽

pp. 1762-1771 ◽

Cited By ~ 7

Author(s):

Rachael H. Earls ◽

Kelly B. Menees ◽

Jaegwon Chung ◽

Claire-Anne Gutekunst ◽

Hyun Joon Lee ◽

...

Keyword(s):

Mouse Model ◽

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Biological Fluids ◽

Lewy Body Dementia ◽

Motor Deficits ◽

Dependent Manner

The pathological hallmark of synucleinopathies, including Lewy body dementia and Parkinson’s disease (PD), is the presence of Lewy bodies, which are primarily composed of intracellular inclusions of misfolded α-synuclein (α-syn) among other proteins. α-Syn is found in extracellular biological fluids in PD patients and has been implicated in modulating immune responses in the central nervous system (CNS) and the periphery. Natural killer (NK) cells are innate effector lymphocytes that are present in the CNS in homeostatic and pathological conditions. NK cell numbers are increased in the blood of PD patients and their activity is associated with disease severity; however, the role of NK cells in the context of α-synucleinopathies has never been explored. Here, we show that human NK cells can efficiently internalize and degrade α-syn aggregates via the endosomal/lysosomal pathway. We demonstrate that α-syn aggregates attenuate NK cell cytotoxicity in a dose-dependent manner and decrease the release of the proinflammatory cytokine, IFN-γ. To address the role of NK cells in PD pathogenesis, NK cell function was investigated in a preformed fibril α-syn–induced mouse PD model. Our studies demonstrate that in vivo depletion of NK cells in a preclinical mouse PD model resulted in exacerbated motor deficits and increased phosphorylated α-syn deposits. Collectively, our data provide a role of NK cells in modulating synuclein pathology and motor symptoms in a preclinical mouse model of PD, which could be developed into a therapeutic for PD and other synucleinopathies.

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