| 101. |
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| 102. |
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| 103. |
- Vanherberghen, Bruno, et al.
(författare)
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Ultrasound-controlled cell aggregation in a multi-well chip
- 2010
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 10:20, s. 2727-2732
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a microplate platform for parallelized manipulation of particles or cells by frequency-modulated ultrasound. The device, consisting of a silicon-glass microchip and a single ultrasonic transducer, enables aggregation, positioning and high-resolution microscopy of cells distributed in an array of 100 microwells centered on the microchip. We characterize the system in terms of temperature control, aggregation and positioning efficiency, and cell viability. We use time-lapse imaging to show that cells continuously exposed to ultrasound are able to divide and remain viable for at least 12 hours inside the device. Thus, the device can be used to induce and maintain aggregation in a parallelized fashion, facilitating long-term microscopy studies of, e.g., cell-cell interactions.
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| 104. |
- Verron, Quentin
(författare)
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Imaging-based methods for NK cell studies at the single-cell level
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The immune system provides defense against infectious agents such as viruses, bacteria and parasites. Besides eliminating extracellular agents, the immune system also constantly monitors our own cells for signs of transformation, including tumor development and virus infection. This process, called immune surveillance, is mediated in part by natural killer (NK) cells. NK cells sense transformation through the interaction of surface receptors with proteins on the surface of the diseased cell. The efficient binding of these receptors results in the formation of a tight contact between the two cells, called an immune synapse. If danger signals dominate in the synapse, the NK cell has the potential to deliver toxic compounds and to bind to specific death receptors at the target cell surface, resulting in the induction of target cell death. Apart from the ability to eliminate transformed cells, NK cells also have an immuno-regulatory function by directly killing other immune cells and by secreting pro- and anti-inflammatory cytokines.Because of these roles, NK cells are of special interest in the growing field of cancer immunotherapy, where the function of immune cells is enhanced to defeat tumor cells. Clinical trials using NK cell-centered therapy have shown promising results against blood-borne cancer, yet progress has been limited against solid tumors. One possible explanation is related to the locally immuno-suppressive environment created by the solid tumor, for which improved research models are necessary. Besides, there is growing evidence of pronounced heterogeneity in the function of individual cells amidst the NK cell pool. Improving our understanding of NK cell biology thus requires advances in dedicated single-cell assays. For this purpose, our research group has previously developed miniaturized multi-well chips where individual cells can be confined and followed by microscopy over periods of several days. Using these microchips, a peculiar group of highly potent NK cells has been identified, which are able to kill several target cells in a row and contribute disproportionately to the overall cytotoxicity, and are therefore referred to as serial-killing NK cells.The work presented in this thesis is focused on developing and applying microscopy-based single-cell assays to the study of NK cell functional heterogeneity, with a particular focus on the mechanistic aspects of cytotoxicity. In Paper I, we investigated the formation and outcome of immune synapses in single cells, using micro-patterning to create distinct spatial distributions of ligands. We observed that synapse formation was guided by the overall shape of the ligands while local signaling regulated the final steps of exocytosis. Paper II is dedicated to the study of the cytotoxic mechanisms used by individual NK cells and their regulation, in particular comparing serial-killing NK cells and moderate killers. Using dedicated fluorescent reporters, we identified a switch between two commonly used killing pathways, degranulation and death ligand engagement, and proposed a model for the underlying process. This topic was further detailed in Paper III, where the contribution of these cytotoxic mechanisms under additional antibody stimulation was studied. The investigation was conducted in a newly developed single-use plastic microchip, designed to enable the generation of multiple simultaneous two- and three-dimensional cell cultures while retaining high imaging performance. In Paper IV, we implemented single-cell retrieval from the silicon-glass microwells. We characterized the performance of our setup and demonstrated its potential at identifying and retrieving rare populations defined by functional readouts. Together, these studies further demonstrate the importance of single-cell analysis in the field of immunology. Besides advancing our understanding of NK cell biology, these developments may prove valuable in developing improved immunotherapies.
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| 105. |
- Verron, Quentin, et al.
(författare)
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Isolation of individual natural killer cells from deep microwell arrays based on functional screening
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Natural killer (NK) cells exhibit functional heterogeneity beyond what is resolved by classical definitions of subpopulations based on cell surface expression of receptors. To develop efficient NK cell-based immunotherapies, we need to understand the parameters that dictate their response to activating and inhibiting stimuli, and environmental factors. For this, new methods are required that can connect NK cell function, e.g. high cytotoxic potential, to molecular or genetic signatures. In this study, we build upon a previously developed microwell chip platform for functional single-cell screening of NK cell behavior, and integrate it into a semi-automated workflow for single-cell identification and isolation. We characterize its performance at retrieving and releasing beads from deep microwells and demonstrate its potential for single NK cell isolation with intact viability. This platform offers new opportunities both for improved understanding of NK cell biology, and for tailoring immunotherapy products with selected NK cell responses.
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| 106. |
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| 107. |
- Verron, Quentin, et al.
(författare)
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NK cells integrate signals over large areas when building immune synapses but require local stimuli for degranulation
- 2021
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 14:684
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Tidskriftsartikel (refereegranskat)abstract
- Immune synapses are large-scale, transient molecular assemblies that serve as platforms for antigen presentation to B and T cells and for target recognition by cytotoxic T cells and natural killer (NK) cells. The formation of an immune synapse is a tightly regulated, stepwise process in which the cytoskeleton, cell surface receptors, and intracellular signaling proteins rearrange into supramolecular activation clusters (SMACs). We generated artificial immune synapses (AIS) consisting of synthetic and natural ligands for the NK cell-activating receptors LFA-1 and CD16 by microcontact printing the ligands into circular-shaped SMAC structures. Live-cell imaging and analysis of fixed human NK cells in this reductionist system showed that the spatial distribution of activating ligands influenced the formation, stability, and outcome of NK cell synapses. Whereas engagement of LFA-1 alone promoted synapse initiation, combined engagement of LFA-1 and CD16 was required for the formation of mature synapses and degranulation. Organizing LFA-1 and CD16 ligands into donut-shaped AIS resulted in fewer long-lasting, symmetrical synapses compared to dot-shaped AIS. NK cells spreading evenly over either AIS shape exhibited similar arrangements of the lytic machinery. However, degranulation only occurred in regions containing ligands that therefore induced local signaling, suggesting the existence of a late checkpoint for degranulation. Our results demonstrate that the spatial organization of ligands in the synapse can affect its outcome, which could be exploited by target cells as an escape mechanism.
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| 108. |
- Walwyn-Brown, Katherine, et al.
(författare)
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Human NK Cells Lyse Th2-Polarizing Dendritic Cells via NKp30 and DNAM-1
- 2018
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 201:7, s. 2028-2041
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Tidskriftsartikel (refereegranskat)abstract
- Cross-talk between NK cells and dendritic cells (DCs) is important in Th1 immune responses, including antitumor immunity and responses to infections. DCs also play a crucial role in polarizing Th2 immunity, but the impact of NK cell-DC interactions in this context remains unknown. In this study, we stimulated human monocyte-derived DCs in vitro with different pathogen-associated molecules: LPS or polyinosinic-polycytidylic acid, which polarize a Th1 response, or soluble egg Ag from the helminth worm Schistosoma mansoni, a potent Th2-inducing Ag. Th2-polarizing DCs were functionally distinguishable from Th1-polarizing DCs, and both showed distinct morphology and dynamics from immature DCs. We then assessed the outcome of autologous NK cells interacting with these differently stimulated DCs. Confocal microscopy showed polarization of the NK cell microtubule organizing center and accumulation of LFA-1 at contacts between NK cells and immature or Th2-polarizing DCs but not Th1-polarizing DCs, indicative of the assembly of an activating immune synapse. Autologous NK cells lysed immature DCs but not DCs treated with LPS or polyinosinic-polycytidylic acid as reported previously. In this study, we demonstrated that NK cells also degranulated in the presence of Th2-polarizing DCs. Moreover, time-lapse live-cell microscopy showed that DCs that had internalized fluorescently labeled soluble egg Ag were efficiently lysed. Ab blockade of NK cell-activating receptors NKp30 or DNAM-1 abrogated NK cell lysis of Th2-polarizing DCs. Thus, these data indicate a previously unrecognized role of NK cell cytotoxicity and NK cell-activating receptors NKp30 and DNAM-1 in restricting the pool of DCs involved in Th2 immune responses.
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| 109. |
- Wiking, Mikaela
(författare)
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Spatial proteome profiling of the compartments of the human cell using an antibody-based approach
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human cell is complex, with countless processes ongoing in parallel in specialized compartments, the organelles. Cells can be studied in vitro by using immortalized cell lines that represent cells in vivo to a varying degree. Gene expression varies between cell types and an average cell line expresses around 10,000-12,000 genes, as measured with RNA sequencing. These genes encode the cell’s proteome; the full set of proteins that perform functions in the cell. In paper I we show that RNA sequencing is a necessary tool for studying the proteome of the human cell.By studying the proteome, and proteins’ localization in the cell, information can be assembled on how the cell functions. Image-based methods allow for detailed spatial resolution of protein localization as well as enable the study of temporal events. Visualization of a protein can be accomplished by using either a cell line that is transfected to express the protein with a fluorescent tag, or by targeting the protein with an affinity reagent such as an antibody. In paper II we present subcellular data for a majority of the human proteins, showing that there is a high degree of complexity in regard to where proteins localize in the cell.Cellular energy is generated in the mitochondria, an important organelle that is also active in many other different functions. Today approximately only a third of the estimated mitochondrial proteome has been validated experimentally, indicating that there is much more to understand with regard to the functions of the mitochondria. In paper III we explore the mitochondrial proteome, based on the results of paper II. We also present a method for sublocalizing proteins to subcompartments that can be performed in a high-throughput manner.To conclude, this thesis shows that transcriptomics is a useful tool for proteome-wide subcellular localization, and presents high-resolution spatial distribution data for the human cell with a deeper analysis of the mitochondrial proteome.
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| 110. |
- Wiklund, Martin, et al.
(författare)
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Acoustofluidics 18 : Microscopy for acoustofluidic micro-devices
- 2012
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 12:18, s. 3221-3234
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Forskningsöversikt (refereegranskat)abstract
- In this tutorial review in the thematic series "Acoustofluidics", we discuss the implementation and practice of optical microscopy in acoustofluidic micro-devices. Examples are given from imaging of acoustophoretic manipulation of particles and cells in microfluidic channels, but most of the discussion is applicable to imaging in any lab-on-a-chip device. The discussion includes basic principles of optical microscopy, different microscopy modes and applications, and design criteria for micro-devices compatible with basic, as well as advanced, optical microscopy.
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