Evaluation of the Functional Capacity of NK Cells of Melanoma Patients in an In Vitro Model of NK Cell Contact with K562 and FemX Tumor Cell Lines

A subpopulation of low density granular lymphocytes that express the natural killer (NK) cell-associated Leu-11 antigen (IgG Fc receptor) were stimulated directly by coculture with an NK-sensitive tumor cell, K562. T lymphocytes (Leu-11-) responded only weakly when cocultured with K562. The response of Leu-11+ cells apparently did not require exogeneous factors or accessory cells. The K562-activated cells retained expression of Leu-11 antigen, acquired activation antigens, and were highly cytotoxic against NK-sensitive and -insensitive tumor cells. Anti-IL-2 receptor monoclonal antibody minimally inhibited the activation of Leu-11+ cells by K562, but completely inhibited the phytohemagglutinin-induced activation of the Leu-11- cells from the same individual. Leu-11+ cells can be divided into Leu-7-11+ and Leu-7+11+ subpopulations using anti-Leu-7 antibody. These subsets were separated by two-color fluorescence-activated cell sorting and cocultured with K562. Proliferation by Leu-7-11+ cells was significantly greater than by Leu-11+7+ cells. Leu-7+11- granular lymphocytes and T lymphocytes (Leu-7-11-) typically proliferated only weakly when cocultured with K562. A proportion of the Leu-7-11+ cells acquired Leu-7 antigen after stimulation with K562, whereas the phenotype of Leu-7+11+, Leu-7+11-, and Leu-7-11- subsets was unaffected. These results demonstrate a developmental relationship between the Leu-7-11+ and Leu-7+11+ lymphocytes and suggest that Leu-7 antigen may be expressed late in the differentiation pathway of NK cells. The direct activation of highly purified Leu-11+ cells by coculture with K562 provides an in vitro model with which to study the activation and maturation of human NK cells.

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CAFs affect the proliferation and treatment response of head and neck cancer spheroids during co-culturing in a unique in vitro model

Cancer Cell International ◽

10.1186/s12935-020-01718-6 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Mustafa Magan ◽

Emilia Wiechec ◽

Karin Roberg

Keyword(s):

Cell Proliferation ◽

Tumor Cell ◽

Head And Neck ◽

Treatment Response ◽

Tumor Cells ◽

In Vitro Model ◽

Tumor Spheroids ◽

Vitro Model ◽

The Impact

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors for which the overall survival rate worldwide is around 60%. The tumor microenvironment, including cancer-associated fibroblasts (CAFs), is believed to affect the treatment response and migration of HNSCC. The aim of this study was to create a biologically relevant HNSCC in vitro model consisting of both tumor cells and CAFs cultured in 3D to establish predictive biomarkers for treatment response, as well as to investigate the impact of CAFs on phenotype, proliferation and treatment response in HNSCC cells. Methods Three different HNSCC patient-derived tumor cell lines were cultured with and without CAFs in a 3D model. Immunohistochemistry of the proliferation marker Ki67, epidermal growth factor receptor (EGFR) and fibronectin and a TUNEL-assay were performed to analyze the effect of CAFs on both tumor cell proliferation and response to cisplatin and cetuximab treatment in tumor spheroids (3D). mRNA expression of epithelial-mesenchymal transition (EMT) and cancer stem cells markers were analyzed using qRT-PCR. Results The results demonstrated increased cell proliferation within the tumor spheroids in the presence of CAFs, correlating with increased expression of EGFR. In spheroids with increased expression of EGFR, a potentiated response to cetuximab treatment was observed. Surprisingly, an increase in Ki67 expressing tumor cells were observed in spheroids treated with cisplatin for 3 days, correlating with increased expression of EGFR. Furthermore, tumor cells co-cultured with CAFs presented an increased EMT phenotype compared to tumor cells cultured alone in 3D. Conclusion Taken together, our results reveal increased cell proliferation and elevated expression of EGFR in HNSCC tumor spheroids in the presence of CAFs. These results, together with the altered EMT phenotype, may influence the response to cetuximab or cisplatin treatment.

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An In Vitro Model for Determining Tumor Cell Migration Under Metabolic Gradients

Advances in Experimental Medicine and Biology - Oxygen Transport to Tissue XL ◽

10.1007/978-3-319-91287-5_32 ◽

2018 ◽

pp. 201-205 ◽

Cited By ~ 2

Author(s):

Yusuke Tsuruno ◽

Kaima Okubo ◽

Takahiro Fujiwara ◽

Yoshihisa Yamaoka ◽

Eiji Takahashi

Keyword(s):

Cell Migration ◽

Tumor Cell ◽

In Vitro Model ◽

Tumor Cell Migration ◽

Vitro Model

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Cytokine-activated NK cells inhibit PMN apoptosis and preserve their functional capacity

Blood ◽

10.1182/blood-2010-01-264903 ◽

2010 ◽

Vol 116 (8) ◽

pp. 1308-1316 ◽

Cited By ~ 35

Author(s):

Nupur Bhatnagar ◽

Henoch S. Hong ◽

Jayendra K. Krishnaswamy ◽

Arash Haghikia ◽

Georg M. Behrens ◽

...

Keyword(s):

Nk Cells ◽

Functional Capacity ◽

Nk Cell ◽

Expression Patterns ◽

Critical Role ◽

Cell Contact ◽

Polymorphonuclear Cells ◽

Soluble Factors ◽

And Function

Abstract Natural killer (NK) cells and polymorphonuclear cells (PMNs) play a critical role in the first line of defense against microorganisms. Upon host infection, PMNs phagocytose invading pathogens with subsequent killing by oxidative or nonoxidative mechanisms. NK cells are known to have immunoregulatory effects on T cells, B cells, dendritic cells (DCs), and monocytes through secretion of various soluble products and cell-cell contact. However, their impact on PMN survival and function is not well known. We found that soluble factors derived from cytokine-activated NK cells delay PMN apoptosis and preserve their ability to perform phagocytosis and produce reactive oxygen species (ROS). The expression patterns of CD11b and CD62L on PMNs differed according to the cytokine combination used for NK-cell stimulation. Irrespective of the NK-cell treatment, however, PMN survival was prolonged with sustained functional capacity. We found that interferon γ, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor α produced by NK cells upon stimulation with cytokines played a crucial role in NK cell–mediated effects on PMNs. Our study demonstrates that soluble factors derived from cytokine-activated NK cells send survival signals to PMNs, which would promote their accumulation and function at the site of inflammation in vivo.

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Impairment of Intramacrophagic Brucella suis Multiplication by Human Natural Killer Cells through a Contact-Dependent Mechanism

Infection and Immunity ◽

10.1128/iai.72.4.2303-2311.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 2303-2311 ◽

Cited By ~ 13

Author(s):

Jacques Dornand ◽

Virginie Lafont ◽

Jane Oliaro ◽

Annie Terraza ◽

Elsa Castaneda-Roldan ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Interleukin 2 ◽

Cell Activity ◽

Human Brucellosis ◽

Cell Contact ◽

Necrosis Factor Alpha ◽

Brucella Suis ◽

Human Natural Killer Cells

ABSTRACT Brucella spp. are facultative intracellular bacteria that can establish themselves and cause chronic disease in humans and animals. NK cells play a key role in host defense. They are implicated in an early immune response to a variety of pathogens. However, it was shown that they do not control Brucella infection in mice. On the other hand, NK cell activity is impaired in patients with acute brucellosis, and recently it was demonstrated that human NK cells mediate the killing of intramacrophagic Mycobacterium tuberculosis in in vitro infection. Therefore, we have analyzed the behavior of Brucella suis infecting isolated human macrophages in the presence of syngeneic NK cells. We show that (i) NK cells impair the intramacrophagic development of B. suis, a phenomenon enhanced by NK cell activators, such as interleukin-2; (ii) NK cells cultured in the presence of infected macrophages are highly activated and secrete gamma interferon and tumor necrosis factor alpha; (iii) impairment of bacterial multiplication inside infected cells is marginally associated with the cytokines produced during the early phase of macrophage-NK cell cocultures; (iv) direct cell-to-cell contact is required for NK cells to mediate the inhibition of B. suis development; and (v) inhibition of B. suis development results from an induction of NK cell cytotoxicity against infected macrophages. Altogether, these findings show that NK cells could participate early in controlling the intramacrophagic development of B. suis in humans. It seems thus reasonable to hypothesize a role for NK cells in the control of human brucellosis. However, by impairing the activity of these cells in the acute phase of the illness, the pathogen should avoid this control.

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Natural-killer cells and dendritic cells: “l'union fait la force”

Blood ◽

10.1182/blood-2005-03-1154 ◽

2005 ◽

Vol 106 (7) ◽

pp. 2252-2258 ◽

Cited By ~ 386

Author(s):

Thierry Walzer ◽

Marc Dalod ◽

Scott H. Robbins ◽

Laurence Zitvogel ◽

Eric Vivier

Keyword(s):

Dendritic Cells ◽

Nk Cells ◽

Natural Killer ◽

Cell Activation ◽

Nk Cell ◽

Interleukin 12 ◽

Cell Contact ◽

Ifn Γ

AbstractSeveral recent publications have focused on the newly described interactions between natural-killer (NK) cells and dendritic cells (DCs). Activated NK cells induce DC maturation either directly or in synergy with suboptimal levels of microbial signals. Immature DCs appear susceptible to autologous NK-cell-mediated cytolysis while mature DCs are protected. NK-cell-induced DC activation is dependent on both tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) secretion and a cell-cell contact involving NKp30. In vitro, interleukin-12 (IL-12)/IL-18, IL-15, and IFN-α/β production by activated DCs enhance, in turn, NK-cell IFN-γ production, proliferation, and cytotoxic potential, respectively. In vivo, NK-cell/DC interactions may occur in lymphoid organs as well as in nonlymphoid tissues, and their consequences are multiple. By inducing DC activation, NK-cell activation induced by tumor cells can indirectly promote antitumoral T-cell responses. Reciprocally, DCs activated through Toll-like receptors (TLRs) induce potent NK-cell activation in antiviral responses. Thus, DCs and NK cells are equipped with complementary sets of receptors that allow the recognition of various pathogenic agents, emphasizing the role of NK-cell/DC crosstalk in the coordination of innate and adaptive immune responses.

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Activation of NK Cells Induces CD4 Expression and Allows HIV−1 Infection.

Blood ◽

10.1182/blood.v108.11.1253.1253 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1253-1253

Author(s):

Hideki Harada ◽

Yumi Goto ◽

Omar F. Dessouki ◽

Shinya Suzu ◽

Seiji Okada

Keyword(s):

T Cells ◽

Hiv Infection ◽

Nk Cells ◽

Nk Cell ◽

Cell Number ◽

Cell Contact ◽

Specific Marker ◽

Activation Markers ◽

Hiv 1

Abstract Natural killer (NK) cells play critical roles in immune surveillance without deliberate prior sensitization and restriction by major histocompatibility complex (MHC). Although function and cell number of NK cells are influenced in AIDS patients, direct interaction between HIV and NK cells is still controversial. Because steady-state NK cells are negative for CD4 which is a key molecule for HIV infection. In this study, we established a condition inducing CD4 expression and HIV-1 infection of NK cells in vitro. CD4 was expressed on NK cells co-cultured with HFWT cells (NK cell-selective stimulating feeder cells) and IL-2. There were no differences in expression level of NK receptors, adhesion molecules and cytotoxicity between CD4+ and CD4- NK cell subpopulations. However, expression of activation markers, CD25 and HLA-DR, and size/granularity of the CD4+ NK cells were higher than CD4- NK cells. CD4+ NK cells expressed co-receptors for HIV-1, CCR5 and CXCR4. Thus, CD4 is induced on NK cells by activation, and the CD4+ NK cells are the possible target for HIV. Next, we exposed HIV-1 clone (JR-FL) to the HFWT-activated NK cells, however, direct HIV infection to the NK cells was not observed. While, co-culture of activated NK cells with HIV infected T cells allowed HIV infection of NK cells. Because NK cell-specific marker, NKp46, was detected on p24+CD3-CD56+ cells but not on CD3+ cells, p24+CD3-CD56+ cells were certainly NK cells. These results demonstrate that NK cells are the targets of HIV. Altogether, our data suggest that CD4+ T cells transfer HIV to NK cells during this cell-to-cell contact, which cause the NK cells to be the long-living viral reservoirs or modify the function of NK cells in HIV-infected patients. Our novel co-culture system using activated NK cells and HIV-infected T cells is the powerful tool to study the function of NK cells on HIV infection.

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Human Natural Killer Cells Mediate Killing of Intracellular Mycobacterium tuberculosis H37Rv via Granule-Independent Mechanisms

Infection and Immunity ◽

10.1128/iai.69.3.1755-1765.2001 ◽

2001 ◽

Vol 69 (3) ◽

pp. 1755-1765 ◽

Cited By ~ 76

Author(s):

Kevin J. Brill ◽

Qing Li ◽

Rhonda Larkin ◽

David H. Canaday ◽

David R. Kaplan ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Human Lymphocytes ◽

Peripheral Blood Lymphocytes ◽

Cell Contact ◽

Mycobacterium Tuberculosis H37rv ◽

Induced Apoptosis

ABSTRACT Despite the continued importance of tuberculosis as a world-wide threat to public health, little is known about the mechanisms used by human lymphocytes to contain and kill the intracellular pathogenMycobacterium tuberculosis. We previously described an in vitro model of infection of human monocytes (MN) with virulent M. tuberculosis strain H37Rv in which the ability of peripheral blood lymphocytes to limit intracellular growth of the organism could be measured. In the current study, we determined that lymphocyte-mediated killing of intracellular M. tuberculosis occurs within the first 24 h of coculture with infected MN. Natural killer (NK) cells isolated from both purified protein derivative (PPD)-positive and PPD-negative subjects were capable of mediating this early killing of intracellular H37Rv. NK cell-mediated killing of intracellular M. tuberculosis was not associated with the production of gamma interferon. Transferred supernatants of cocultured NK cells and M. tuberculosis-infected MN could not mediate the killing of intracellular M. tuberculosis, and Transwell studies indicated that direct cell-to-cell contact was required for NK cells to mediate the killing of the organism. Killing was not dependent upon exocytosis of NK cell cytotoxic granules. NK cells induced apoptosis of mycobacterium-infected MN, but neither killing of intracellularM. tuberculosis by NK cells nor NK cell-induced apoptosis of infected MN was inhibited by blocking the interaction of FasL and Fas. Thus, human NK cells may mediate killing of intracellular M. tuberculosis via alternative apoptotic pathways.

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