| 1. |
- Prager, Isabel, et al.
(författare)
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NK cells switch from granzyme B to death receptor–mediated cytotoxicity during serial killing
- 2019
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 7:9, s. 2113-2127
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Tidskriftsartikel (refereegranskat)abstract
- NK cells eliminate virus-infected and tumor cells by releasing cytotoxic granules containing granzyme B (GrzB) or by engaging death receptors that initiate caspase cascades. The orchestrated interplay between both cell death pathways remains poorly defined. Here we simultaneously measure the activities of GrzB and caspase-8 in tumor cells upon contact with human NK cells. We observed that NK cells switch from inducing a fast GrzB-mediated cell death in their first killing events to a slow death receptor–mediated killing during subsequent tumor cell encounters. Target cell contact reduced intracellular GrzB and perforin and increased surface-CD95L in NK cells over time, showing how the switch in cytotoxicity pathways is controlled. Without perforin, NK cells were unable to perform GrzB-mediated serial killing and only killed once via death receptors. In contrast, the absence of CD95 on tumor targets did not impair GrzB-mediated serial killing. This demonstrates that GrzB and death receptor–mediated cytotoxicity are differentially regulated during NK cell serial killing.
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| 2. |
- Sandoz, Patrick, et al.
(författare)
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Modulation of lytic molecules restrain serial killing in γδ T lymphocytes
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies.
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| 3. |
- Sandström, Niklas, 1981-, et al.
(författare)
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Miniaturized and multiplexed high-content screening of drug and immune sensitivity in a multichambered microwell chip
- 2022
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Ingår i: CELL REPORTS METHODS. - : Elsevier BV. - 2667-2375. ; 2:7
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Tidskriftsartikel (refereegranskat)abstract
- Here, we present a methodology based on multiplexed fluorescence screening of two-or three-dimensional cell cultures in a newly designed multichambered microwell chip, allowing direct assessment of drug or im-mune cell cytotoxic efficacy. We establish a framework for cell culture, formation of tumor spheroids, fluores-cence labeling, and imaging of fixed or live cells at various magnifications directly in the chip together with data analysis and interpretation. The methodology is demonstrated by drug cytotoxicity screening using ovarian and non-small cell lung cancer cells and by cellular cytotoxicity screening targeting tumor spheroids of renal carcinoma and ovarian carcinoma with natural killer cells from healthy donors. The miniaturized format allowing long-term cell culture, efficient screening, and high-quality imaging of small sample volumes makes this methodology promising for individualized cytotoxicity tests for precision medicine.
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| 4. |
- van Ooijen, Hanna, et al.
(författare)
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Distinct mechanistic responses in serial-killing NK cells during natural and antibody-dependent cytotoxicity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Natural Killer (NK) cells are potent cytotoxic immune cells, yet the population exhibits a large phenotypical and functional heterogeneity. Here we investigated the mechanisms underlying NK cell serial killing during natural and antibody-dependent cellular cytotoxicity (ADCC). We leveraged on our microwell chip platform to relate cellular function with organelle content and intracellular protein expression, in individual NK cells. We observed that highly potent serial-killing NK cells more often deployed degranulation and induced necrosis, and that they were multifunctional, producing INF-γ and TNF-α. We probed for intrinsic differences between NK cells that performed serial killing compared to other NK cells, yet found no distinction in the migration dynamics, nor in the lysosomal and mitochondrial load of the cells. However, when measuring the calcium signaling of the cells in consecutive interactions, we revealed that serial-killing NK cells were able to maintain calcium signaling longer than cells that interrupted their killing sequence. By staining the cells at the end of the cytotoxicity assay, we found that NK cells that had stopped killing still showed clear intracellular expression of granzyme B-positive granules. Thus, it appears that decreased signaling rather than depletion of lytic cargo is the reason for NK cell exhaustion in our assay. Our results motivate exploring ways to enhance the signaling machinery in NK cells to achieve sustained functional responses.
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| 5. |
- van Ooijen, Hanna, et al.
(författare)
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Screening and high-resolution imaging of dynamic single-cell responses in 2D and 3D using a novel disposable microwell chip
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Single-cell assays are developed as novel tools for the identification and characterization of individual cell responses. Some methods provide snapshots of the phenotype of the cell, such as flow cytometry and single-cell RNA sequencing, whereas others, almost exclusively microscopy-based, can be used for longitudinal studies of individual cells. In the present study, we developed an easy-to-use, disposable, thermoplastic microwell chip, designed to support screening and high-resolution imaging of single-cell behavior in two-and three-dimensional cell cultures. We confirm its excellent optical properties and provide simple protocols for efficient long-term cell culture of suspension and adherent cells, the latter grown either as monolayers or as hundreds of single, uniformly-sized spheroids. We demonstrate the applicability of the system for single-cell analysis by correlating the dynamic cytotoxic response of single immune cells to their intracellular cytolytic load at the end of the assay. Additionally, we illustrate highly multiplex cytotoxicity screening of tumor spheroids in the chip, comparing the effect of environment cues characteristic of the tumor microenvironment on NK cell-induced killing. Following the functional screening, we perform high-resolution 3D immunofluorescent imaging of infiltrating NK cells within the spheroid volumes.
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| 6. |
- 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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| 7. |
- 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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| 8. |
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| 9. |
- 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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| 10. |
- Wernersson, Erik, et al.
(författare)
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Deconwolf enables high-performance deconvolution of widefield fluorescence microscopy images
- 2024
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Ingår i: Nature Methods. - 1548-7091 .- 1548-7105. ; 21:7, s. 1245-1256
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Tidskriftsartikel (refereegranskat)abstract
- Microscopy-based spatially resolved omic methods are transforming the life sciences. However, these methods rely on high numerical aperture objectives and cannot resolve crowded molecular targets, limiting the amount of extractable biological information. To overcome these limitations, here we develop Deconwolf, an open-source, user-friendly software for high-performance deconvolution of widefield fluorescence microscopy images, which efficiently runs on laptop computers. Deconwolf enables accurate quantification of crowded diffraction limited fluorescence dots in DNA and RNA fluorescence in situ hybridization images and allows robust detection of individual transcripts in tissue sections imaged with ×20 air objectives. Deconvolution of in situ spatial transcriptomics images with Deconwolf increased the number of transcripts identified more than threefold, while the application of Deconwolf to images obtained by fluorescence in situ sequencing of barcoded Oligopaint probes drastically improved chromosome tracing. Deconwolf greatly facilitates the use of deconvolution in many bioimaging applications.
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