High-efficient lentiviral vector-mediated gene transfer into primary human NK cells

Micucci, Federica; Zingoni, Alessandra; Piccoli, Mario; Frati, Luigi; Santoni, Angela; Galandrini, Ricciarda

doi:10.1016/j.exphem.2006.06.001

Cited by 45 publications

(50 citation statements)

References 36 publications

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“…[21][22][23] By means of lentiviral-driven expression, we obtained high transduction efficiency without affecting NK-cell phenotype and functional properties. 25 As illustrated in Figure 1A, in isolated NK cells GFP-PH distributed throughout the plasma membrane where it localized in discrete microdomains; as expected, control GFP distributed homogeneously within the cell ( Figure 1B). To monitor PIP2 dynamics in live cells during the cytolytic event, anti-CD16-treated NK cells were allowed to bind to Fc receptor-positive P815 target cells by reverse antibodydependent cellular cytotoxicity (rADCC) and analyzed by timelapse confocal microscopy.…”

Section: Pip2 Dynamics During the Activation Of Nk Cytolytic Machinerymentioning

confidence: 89%

“…[21][22][23] By means of lentiviral-driven expression, we obtained high transduction efficiency without affecting NK-cell phenotype and functional properties. 25 As illustrated in Figure 1A, For personal use only. on April 7, 2019. by guest www.bloodjournal.org From in isolated NK cells GFP-PH distributed throughout the plasma membrane where it localized in discrete microdomains; as expected, control GFP distributed homogeneously within the cell ( Figure 1B).…”

Section: Pip2 Dynamics During the Activation Of Nk Cytolytic Machinerymentioning

confidence: 96%

“…25 shRNA-encoding DNA sequences targeting PI5KI␣, PI5KI␤, and PI5KI␥ 18,26 were cloned into XhoI/HpaI site of lentiviral pLL3.7 vector. 27…”

Section: Lentiviral Vector Cloning and Productionmentioning

confidence: 99%

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PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

Micucci

Capuano

Marchetti

et al. 2008

Blood

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Although membrane phospholipid phosphatidylinositol-4,5bisphosphate (PIP2) plays a key role as signaling intermediate and coordinator of actin dynamics and vesicle trafficking, it remains completely unknown its involvement in the activation of cytolytic machinery. By live confocal imaging of primary human natural killer (NK) cells expressing the chimeric protein GFP-PH, we observed, during effector-target cell interaction, the consumption of a preexisting PIP2 pool, which is critically required for the activation of cytolytic machinery. We identified type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) ␣ and ␥ isoforms as the enzymes responsible for PIP2 synthesis in NK cells. By hRNA-driven gene silencing, we observed that both enzymes are required for the proper activation of NK cytotoxicity and for inositol-1,4,5-trisphosphate (IP3) generation on receptor stimulation. In an attempt to elucidate the specific step controlled by PI5KIs, we found that lytic granule secretion but not polarization resulted in impaired PI5KI␣-and PI5KI␥-silenced cells. Our findings delineate a novel mechanism implicating PI5KI␣ and PI5KI␥ isoforms in the synthesis of PIP2 pools critically required for IP3-dependent Ca 2؉ IntroductionNatural killer (NK) cells and cytotoxic T lymphocytes are critical effectors in the defense against tumor and viral infections 1 ; they exert cytotoxic function through the polarized secretion of granules containing proteolytic molecules, such as perforin and granzymes. This process involves several steps, including the formation of a cytolytic synapse between cytolytic effector and target cell, the rapid reorientation of the microtubule-organizing center along with lytic granules toward the target contact area followed by granule docking and fusion at specialized secretory domains within the cytolytic synapse. 2,3 Several structurally distinct receptors have been implicated in the activation of NK-cell cytolytic machinery: when cross-linked by the corresponding ligands on target cell, they trigger multiple and intersecting signaling pathways responsible for functional activation. 4 A vast array of activating NK receptors belonging to different families are coupled to the lipid modifying enzymes phosphatidylinositol3-kinase (PI3K) and phospholipase C␥ (PLC␥), which provide signals critically required for the activation of the cytolytic machinery [5][6][7][8] ; notably, both enzymes use the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) as common substrate. 9 Besides its role as signaling intermediate, PIP2 also acts as critical regulator of various cellular processes, including actin remodeling, membrane and vesicle trafficking, adhesion, and ion transport. 10,11 Surprisingly, the role of PIP2 and its regulatory mechanisms in lymphocyte-mediated cytotoxicity remain completely undefined.The main cellular source of PIP2 are type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) family members which phosphorylate PI4P on the D5 position of the inositol ring. Three major PI5KI is...

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Section: Pip2 Dynamics During the Activation Of Nk Cytolytic Machinerymentioning

confidence: 89%

“…[21][22][23] By means of lentiviral-driven expression, we obtained high transduction efficiency without affecting NK-cell phenotype and functional properties. 25 As illustrated in Figure 1A, For personal use only. on April 7, 2019. by guest www.bloodjournal.org From in isolated NK cells GFP-PH distributed throughout the plasma membrane where it localized in discrete microdomains; as expected, control GFP distributed homogeneously within the cell ( Figure 1B).…”

Section: Pip2 Dynamics During the Activation Of Nk Cytolytic Machinerymentioning

confidence: 96%

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PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

Micucci

Capuano

Marchetti

et al. 2008

Blood

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“…Degranulation assay and IFN-g production NK cell-mediated cytotoxicity was evaluated using the degranulation lysosomal marker CD107a as described previously (3,23). As source of effector cells, freshly isolated NK cells were used (24).…”

Section: Analysis and Isolation Of Senescent Cellsmentioning

confidence: 99%

Reactive Oxygen Species– and DNA Damage Response–Dependent NK Cell Activating Ligand Upregulation Occurs at Transcriptional Levels and Requires the Transcriptional Factor E2F1

Soriani

Iannitto

Ricci

et al. 2014

The Journal of Immunology

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Increasing evidence indicates that cancer cell stress induced by chemotherapeutic agents promote antitumor immune responses and contribute to their full clinical efficacy. In this article, we identify the signaling events underlying chemotherapy-induced NKG2D and DNAM-1 ligand expression on multiple myeloma (MM) cells. Our findings indicate that sublethal doses of doxorubicin and melphalan initiate a DNA damage response (DDR) controlling ligand upregulation on MM cell lines and patient-derived malignant plasma cells in Chk1/2-dependent and p53-independent manner. Drug-induced MICA and PVR gene expression are transcriptionally regulated and involve DDR-dependent E2F1 transcription factor activity. We also describe the involvement of changes in the redox state in the control of DDR-dependent upregulation of ligand surface expression and gene transcriptional activity by using the antioxidant agent N-acetyl-l-cysteine. Finally, in accordance with much evidence indicating that DDR and oxidative stress are major determinants of cellular senescence, we found that redox-dependent DDR activation upon chemotherapeutic treatment is critical for MM cell entry in premature senescence and is required for the preferential ligand upregulation on senescent cells, which are preferentially killed by NK cells and trigger potent IFN-γ production. We propose immunogenic senescence as a mechanism that promotes the clearance of drug-treated tumor cells by innate effector lymphocytes, including NK cells.

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“…The integrity of all constructs was verified by automated sequencing. The lentivirus infection of NK92 cells was performed using a previously described protocol (40). NK92/luciferase-shRNAVav1-GFP cells and NK92/Grb2-shRNA-Vav1-GFP cells expressing the same level of Vav1-GFP were sorted 7 to 10 days later on a Beckton Dickinson FACSVantage SE flow cytometer at the Flow Cytometry Core Facility, Department of Pathology and Immunology, Washington University, St. Louis, MO.…”

Section: Cells Lines and Antibodies Human Interleukin-2 (Il-2)-depenmentioning

confidence: 99%

Phosphatidylinositol 3-Kinase Activation Is Required To Form the NKG2D Immunological Synapse

Giurisato

Cella

Takai

et al. 2007

Molecular and Cellular Biology

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The receptor NKG2D allows natural killer (NK) cells to detect virally infected, stressed, and tumor cells. In human cells, NKG2D signaling is mediated through the associated DAP10 adapter. Here we show that engagement of NKG2D by itself is sufficient to stimulate the formation of the NK immunological synapse (NKIS), with recruitment of NKG2D to the center synapse. Mutagenesis studies of DAP10 revealed that the phosphatidylinositol 3-kinase binding site, but not the Grb2 binding site, was required and sufficient for recruitment of DAP10 to the NKIS. Surprisingly, we found that in the absence of the Grb2 binding site, Grb2 was still recruited to the NKIS. Since the recruitment of Grb2 was dependent on phosphatidylinositol-(3,4,5)-trisphosphate (PIP 3 ), we explored the possibility that recruitment to the NKIS is mediated by a pleckstrin homology (PH) domain-containing binding partner for Grb2. We found that the PH domain of SOS1, but not that of Vav1, was able to be recruited by PIP 3 . These results provide new insights into the mechanism of immunological synapse formation and also demonstrate how multiple mechanisms can be used to recruit the same signaling proteins to the plasma membrane.

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High-efficient lentiviral vector-mediated gene transfer into primary human NK cells

Cited by 45 publications

References 36 publications

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

Reactive Oxygen Species– and DNA Damage Response–Dependent NK Cell Activating Ligand Upregulation Occurs at Transcriptional Levels and Requires the Transcriptional Factor E2F1

Phosphatidylinositol 3-Kinase Activation Is Required To Form the NKG2D Immunological Synapse

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