| 1. |
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| 2. |
- Forslund, E., et al.
(författare)
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Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations
- 2012
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 3:OCT, s. 300-
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Tidskriftsartikel (refereegranskat)abstract
- Each individual has a heterogeneous pool of NK cells consisting of cells that may be specialized towards specific functional responses such as secretion of cytokines or killing of tumor cells. Many conventional methods are not fit to characterize heterogeneous populations as they measure the average response of all cells. Thus, there is a need for experimental platforms that provide single cell resolution. In addition, there are transient and stochastic variations in functional responses at the single cell level, calling for methods that allow studies of many events over extended periods of time. This paper presents a versatile microchip platform enabling long-term microscopic studies of individual NK cells interacting with target cells. Each microchip contains an array of microwells, optimized for medium or high-resolution time-lapse imaging of single or multiple NK and target cells, or for screening of thousands of isolated NK-target cell interactions. Individual NK cells confined with target cells in small microwells is a suitable setup for high-content screening and rapid assessment of heterogeneity within populations, while microwells of larger dimensions are appropriate for studies of NK cell migration and sequential interactions with multiple target cells. By combining the chip technology with ultrasonic manipulation, NK and target cells can be forced to interact and positioned with high spatial accuracy within individual microwells.This setup effectively and synchronously creates NK-target conjugates at hundreds of parallel positions in the microchip. Thus, this facilitates assessment of temporal aspects of NK-target cell interactions, e.g., conjugation, immune synapse formation, and cytotoxic events.The microchip platform presented here can be used to effectively address questions related to fundamental functions of NK cells that can lead to better understanding of how the behavior of individual cells add up to give a functional response at the population level.
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| 3. |
- Frisk, Thomas, et al.
(författare)
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A silicon-glass microwell platform for high-resolution imaging and high-content screening with single cell resolution
- 2011
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Ingår i: Biomedical microdevices (Print). - : Springer Science and Business Media LLC. - 1387-2176 .- 1572-8781. ; 13:4, s. 683-693
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Tidskriftsartikel (refereegranskat)abstract
- We present a novel microwell array platform suited for various cell-imaging assays where single cell resolution is important. The platform consists of an exchangeable silicon-glass microchip for cell biological applications and a custom made holder that fits in conventional microscopes. The microchips presented here contain arrays of miniature wells, where the well sizes and layout have been designed for different applications, including single cell imaging, studies of cell-cell interactions or ultrasonic manipulation of cells. The device has been designed to be easy to use, to allow long-term assays (spanning several days) with read-outs based on high-resolution imaging or high-content screening. This study is focused on screening applications and an automatic cell counting protocol is described and evaluated. Finally, we have tested the device and automatic counting by studying the selective survival and clonal expansion of 721.221 B cells transfected to express HLA Cw6-GFP compared to untransfected 721.221 B cells when grown under antibiotic selection for 3 days. The device and automated analysis protocol make up the foundation for development of several novel cellular imaging assays.
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| 4. |
- Frisk, T., et al.
(författare)
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Live-cell imaging of natural killer cell mediated tumor rejection in arrays of microwells
- 2010
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Ingår i: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010. - 9781618390622 ; , s. 950-952
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Konferensbidrag (refereegranskat)abstract
- Due to the inherent heterogeneity of immune cell populations a comprehensive view of immune functions can only be achieved by collecting data from many individual cells. We here demonstrate a novel multi-well microchip platform for cell analysis, which we use to study the interaction between natural killer (NK) cells and their target cells.
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| 5. |
- Guldevall, Karolin
(författare)
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Development of Microchip-based Assays to Study Immune Cell Interactions at the Single Cell Level
- 2011
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Immune cell populations are constantly divided into smaller and smaller subsets defined by newly emerging cellular markers. However, there is a growing awareness of the functional heterogeneities in between cells even within small populations, in addition to the heterogeneity over time. One may ask whether a population is correctly defined only by cellular markers or if the functionality should be regarded as well? Many of today’s techniques only measures at the population level, giving an average estimate of the behavior of that pool of cells, but failing to detect rare possibly important events. Thus, high-throughput experimental approaches to analyze single cells over time are required to address cellular heterogeneity. Progress in the fields of microfabrication, microscopy and computing have paved the way for increasingly efficient tools for studies on the single cell level, and a variety of devices have been described by others. However, few of them are suitable for long-term imaging of dynamic events such as cell-cell interactions or migration. In addition, for efficient recording of many individual events it is desirable to scale down the cells’ interaction volume; not only to shorten the time to interaction, but also to increase the number of individual events in a given area; thereby pushing a screening approach. To address these questions, a complete microwell array system for imaging of immune cell responses with single-cell resolution was designed. The platform consists of a range of silicon-glass microchips with arrays of miniature wells for incubation of cells and a custom made holder that fits conventional microscopes. The device has been designed to allow cells to be kept viable for several days in the wells, to be easy to use and to allow high-resolution imaging. Five different designs were fabricated; all with a specific type of assay in mind, and were evaluated regarding biocompatibility and functionality. One design is aimed towards screening applications, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated; this program is also described and evaluated. We here show that our silicon microwell platform allows long-term studies (up to several days), with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using time-lapse imaging we confirmed immune cell heterogeneity in NK cell populations regarding both cytotoxicity and migrational behavior. The automatic counting program was tested and showed similar results compared to both manual counting and FACS. In addition, the large numbers of wells that can be simultaneously imaged, provide new statistical information that will lead to a better understanding of the function and regulation of the immune system at the single cell level. Altogether, our technique enables novel types of cellular imaging assays allowing data collection at a level of resolution not previously obtained – this was shown to be important for performing basic cell biological studies, but may also prove valuable in the proposed future medical applications.
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| 6. |
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| 7. |
- Guldevall, Karolin, et al.
(författare)
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Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:11, s. e15453-
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Tidskriftsartikel (refereegranskat)abstract
- New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.
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| 8. |
- Guldevall, Karolin, 1981-, et al.
(författare)
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Microchip screening platform for assessment of cytotoxic effector cells
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) or T cells within larger populations. Human primary NK cells or human Epstein-Barr virus (EBV)- specific T cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of effector and live or dead target cells in each well could be assessed at different time-points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic to the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12 hours long experiment. We demonstrate that this assay can be used to enumerate and characterize cytotoxic cells, something that could find clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.
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| 9. |
- Guldevall, Karolin, et al.
(författare)
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Microchip screening Platform for single cell assessment of NK cell cytotoxicity
- 2016
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Ingår i: Frontiers in Immunology. - : FRONTIERS MEDIA SA. - 1664-3224. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (approximate to 75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (>= 3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.
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| 10. |
- Guldevall, Karolin, 1981-
(författare)
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Single Cell Investigations of the Functional Heterogeneity Within Immune Cell Populations : a Microchip-based Study
- 2014
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Immune cell populations are constantly divided into smaller and smaller subsets defined by newly emerging cellular markers. However, there is a growing awareness of the functional heterogeneities in between cells even within small populations, in addition to the heterogeneity over time. One may ask whether a population is correctly defined only by cellular markers or if the functionality should be regarded as well? Many of today’s techniques only measure at the population level, giving an average estimate of the behavior of that pool of cells, but failing to detect rare possibly important events. Thus, high-throughput experimental approaches to analyze single cells over time are required to address cellular heterogeneity.Progress in the fields of microfabrication, microscopy and computing have paved the way for increasingly efficient tools for studies on the single cell level, and a variety of devices have been described by others. However, few of them are suitable for long-term imaging of dynamic events such as cell-cell interactions or migration. In addition, for efficient recording of many individual events it is desirable to scale down the cells’ interaction volume; not only to shorten the time to interaction, but also to increase the number of individual events in a given area; thereby pushing a screening approach.To address these questions, a complete microwell array system for imaging of immune cell responses with single-cell resolution was designed. The platform consists of a range of silicon-glass microchips with arrays of miniature wells for incubation of cells and a custom made holder that fits conventional microscopes. The device has been designed to allow cells to be kept viable for several days in the wells, to be easy to use and to allow high-resolution imaging. Five different designs were fabricated; all with a specific type of assay in mind, and were evaluated regarding biocompatibility and functionality. Here, the design aimed for screening applications is the main focus. In this approach a large amount, tens of thousands, of small wells are imaged two to three times: first directly post-seeding of effector and target cells to register the well’s content, and second after some time has passed to allow for cell-cell interactions. The final read-out is the number of killed target cells in each well, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated.We here show that our silicon microwell platform allows long-term studies with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using both time-lapse imaging and the screening approach we confirmed and investigated immune cell heterogeneity within NK cell populations in regards to both cytotoxicity and migrational behavior. In addition, two different types of cytolytic behavior in NK cells, termed fast and slow killing, were described and evaluated in regards to dynamic parameters; like conjugation and attachment time. We could also quantify the type of cytolytic response in relation to serial killing NK cells, and saw that serial killing NK cells more often induced fast target cell death. Further investigations using the screening approach have shown that serial killing NK cells also differ from other NK cells in their morphology, being both larger and with a more elongated shape. So far the platform has been used to gain better understanding of some aspects of NK cell biology, but there is still much left to explore. With the addition of an automatic counting program, the large numbers of wells that can be simultaneously imaged will provide new statistical information and enable higher throughput.Altogether, our family of techniques enables novel types of cellular imaging assays allowing data collection at a level of resolution not previously obtained – this was shown to be important for performing basic cell biological studies, but may also prove valuable in the proposed future medical applications such as adoptive cell therapy and stem cell transplantation.
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| 11. |
- Sandström, Niklas, 1981-, et al.
(författare)
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Laser-induced deep etching of glass for live cell assays
- 2021
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Ingår i: MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 579-580
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Konferensbidrag (refereegranskat)abstract
- Glass materials have excellent optical and chemical properties for microscopy-based live cell assays but state-of-the-art methods for microfabrication of Lab-on-Chip (LoC) devices are often limited by either complex manufacturing and/or low quality results. In this work, we have evaluated glass microwell array chips produced using a recently introduced laser-based microfabrication method. Three different types of microwell designs have been tested for imaging and screening of on-chip cell cultures and live cell assays.
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| 12. |
- Sandström, Niklas, 1981-, et al.
(författare)
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Live single cell imaging assays in glass microwells produced by laser-induced deep etching
- 2022
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 22:11, s. 2107-2121
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Tidskriftsartikel (refereegranskat)abstract
- Miniaturization of cell culture substrates enables controlled analysis of living cells in confined micro-scale environments. This is particularly suitable for imaging individual cells over time, as they can be monitored without escaping the imaging field-of-view (FoV). Glass materials are ideal for most microscopy applications. However, with current methods used in life sciences, glass microfabrication is limited in terms of either freedom of design, quality, or throughput. In this work, we introduce laser-induced deep etching (LIDE) as a method for producing glass microwell arrays for live single cell imaging assays. We demonstrate novel microwell arrays with deep, high-aspect ratio wells that have rounded, dimpled or flat bottom profiles in either single-layer or double-layer glass chips. The microwells are evaluated for microscopy-based analysis of long-term cell culture, clonal expansion, laterally organized cell seeding, subcellular mechanics during migration and immune cell cytotoxicity assays of both adherent and suspension cells. It is shown that all types of microwells can support viable cell cultures and imaging with single cell resolution, and we highlight specific benefits of each microwell design for different applications. We believe that high-quality glass microwell arrays enabled by LIDE provide a great option for high-content and high-resolution imaging-based live cell assays with a broad range of potential applications within life sciences.
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| 13. |
- Sarha, Dhifaf, et al.
(författare)
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161533 TriKE stimulates NK-cell function to overcome myeloid-derived suppressor cells in MDS
- 2018
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Ingår i: Blood Advances. - : AMER SOC HEMATOLOGY. - 2473-9529 .- 2473-9537. ; 2:12, s. 1459-1469
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Tidskriftsartikel (refereegranskat)abstract
- Myelodysplastic syndrome (MDS) is a clonal heterogeneous stem cell disorder driven by multiple genetic and epigenetic alterations resulting in ineffective hematopoiesis. MDS has a high frequency of immune suppressors, including myeloid-derived suppressor cells (MDSCs), that collectively result in a poor immune response. MDSCs in MDS patients express CD155 that ligates the T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) and delivers an inhibitory signal to natural killer (NK) cells. To mediate a productive immune response against MDS, negative regulatory checkpoints, like TIGIT, expressed on MDS NK cells must be overcome. NK cells can be directed to lyse MDS cells by bispecific killer engagers (BiKEs) that ligate CD16 on NK cells and CD33 on MDS cells. However, such CD16 x CD33 (1633) BiKEs do not induce the proliferative response in MDS NK cells needed to sustain their function. Here, we show that the addition of an NK stimulatory cytokine, interleukin-15 (IL-15), into the BiKE platform leads to productive IL-15 signaling without TIGIT upregulation on NK cells from MDS patients. Lower TIGIT expression allowed NK cells to resist MDSC inhibition. When compared with 1633 BiKE, 161533 trispecific killer engager (TriKE)-treated NK cells demonstrated superior killing kinetics associated with increased STAT5 phosphorylation. Furthermore, 161533 TriKE-treated MDS NK cells had higher proliferation and enhanced NK-cell function than 1633 BiKE-treated cells without the IL-15 linker. Collectively, our data demonstrate novel characteristics of the 161533 TriKE that support its application as an immunotherapeutic agent for MDS patients.
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| 14. |
- Srpan, Katja, et al.
(författare)
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Shedding of CD16 disassembles the NK cell immune synapse and boosts serial engagement of target cells
- 2018
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 217:9, s. 3267-3283
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Tidskriftsartikel (refereegranskat)abstract
- Natural Killer (NK) cells can engage multiple virally infected or tumor cells sequentially and deliver perforin for cytolytic killing of these targets. Using microscopy to visualize degranulation from individual NK cells, we found that repeated activation via the Fc receptor CD16 decreased the amount of perforin secreted. However, perforin secretion was restored upon subsequent activation via a different activating receptor, NKG2D. Repeated stimulation via NKG2D also decreased perforin secretion, but this was not rescued by stimulation via CD16. These different outcomes of sequential stimulation could be accounted for by shedding of CD16 being triggered by cellular activation. The use of pharmacological inhibitors and NK cells transfected to express a noncleavable form of CD16 revealed that CD16 shedding also increased NK cell motility and facilitated detachment of NK cells from target cells. Disassembly of the immune synapse caused by CD16 shedding aided NK cell survival and boosted serial engagement of target cells. Thus, counterintuitively, shedding of CD16 may positively impact immune responses.
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| 15. |
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| 16. |
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| 17. |
- Van der Meer, J. M. R., et al.
(författare)
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IL-15 superagonist N-803 improves IFNγ production and killing of leukemia and ovarian cancer cells by CD34+ progenitor-derived NK cells
- 2020
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Ingår i: Cancer Immunology and Immunotherapy. - : Springer Science and Business Media Deutschland GmbH. - 0340-7004 .- 1432-0851.
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Tidskriftsartikel (refereegranskat)abstract
- Allogeneic natural killer (NK) cell transfer is a potential immunotherapy to eliminate and control cancer. A promising source are CD34 + hematopoietic progenitor cells (HPCs), since large numbers of cytotoxic NK cells can be generated. Effective boosting of NK cell function can be achieved by interleukin (IL)-15. However, its in vivo half-life is short and potent trans-presentation by IL-15 receptor α (IL-15Rα) is absent. Therefore, ImmunityBio developed IL-15 superagonist N-803, which combines IL-15 with an activating mutation, an IL-15Rα sushi domain for trans-presentation, and IgG1-Fc for increased half-life. Here, we investigated whether and how N-803 improves HPC-NK cell functionality in leukemia and ovarian cancer (OC) models in vitro and in vivo in OC-bearing immunodeficient mice. We used flow cytometry-based assays, enzyme-linked immunosorbent assay, microscopy-based serial killing assays, and bioluminescence imaging, for in vitro and in vivo experiments. N-803 increased HPC-NK cell proliferation and interferon (IFN)γ production. On leukemia cells, co-culture with HPC-NK cells and N-803 increased ICAM-1 expression. Furthermore, N-803 improved HPC-NK cell-mediated (serial) leukemia killing. Treating OC spheroids with HPC-NK cells and N-803 increased IFNγ-induced CXCL10 secretion, and target killing after prolonged exposure. In immunodeficient mice bearing human OC, N-803 supported HPC-NK cell persistence in combination with total human immunoglobulins to prevent Fc-mediated HPC-NK cell depletion. Moreover, this combination treatment decreased tumor growth. In conclusion, N-803 is a promising IL-15-based compound that boosts HPC-NK cell expansion and functionality in vitro and in vivo. Adding N-803 to HPC-NK cell therapy could improve cancer immunotherapy.
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| 18. |
- van Ooijen, Hanna
(författare)
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Mechanisms regulating Natural Killer Cell Cytotoxicity
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Over the last 50 years, cancer survival rates have steadily improved thanks to earlier detection and novel treatment regimens. For example, drugs that act on the immune system, so-called immunotherapy, have drastically increased the prospect of survival for patients suffering from some of the most aggressive cancer types including advanced malignant melanoma. Nevertheless, many patients still do not respond to neither traditional treatments or immunotherapy. Expanding the knowledge of immune cell function, and of how the immune system is dysregulated in cancer, will hence be fundamental for the development of more efficient treatment strategies.Natural Killer (NK) cells, a type of cytotoxic innate immune cell, have been identified as a target for immunotherapy due to their capacity to recognize and destroy cancer cells. Tumor-infiltrating NK cells often display reduced functionality, and therapies targeting NK cells therefore aim at enhancing their anti-tumor activity. However, the responses of individual NK cells are highly heterogeneous, and although some cells efficiently destroy harmful targets, others do not.Both functionality and phenotype are most efficiently studied using single-cell approaches, as these can resolve differences within heterogenous populations. In immunology, flow cytometry has been the golden standard for single-cell assessment, but this method has limited applicability when studying dynamic processes. For this purpose, imaging-based methods are a superior alternative, especially when combined with spatial confinement of single cells.In this thesis, I describe the development and application of microscopy-based approaches for the study of single NK cell functional responses. Specifically, I have sought to increase our understanding of the mechanisms regulating NK cell cytotoxicity. In Paper 1, we developed a plastic microwell chip for the investigation of the function of NK cells at the single-cell level. The platform was used to examine how NK cell cytotoxicity is negatively affected by several factors present in the tumor microenvironment, including limited glucose and glutamine availability. In Paper 2, we explored the cytotoxic mechanisms deployed by individual NK cells during sequential killing. We showed that single NK cells switch from almost exclusively applying degranulation in early killing events, to using death ligand engagement for their final kill. In Paper 3, we further investigated the factors prohibiting NK cells from continued killing, during both natural ligand and antibody-mediated cytotoxicity. We discovered that most NK cells retain a large pool of granzyme B-positive lytic granules after they have ceased killing, but calcium signaling is maintained only in NK cells that are capable of sequential killing. In Paper 4, we explored ways of improving the therapeutic efficacy of in vitro-activated immune cells by avoiding their rejection by the host immune system. We showed that the combined deletion of CD54 and CD58 in grafted cells resulted in a reduced recognition by host NK cells, thereby improving graft survival.In summary, the work presented in this thesis demonstrates the importance of single-cell methods for characterizing immune cell function. Such advancements are crucial for the continued development of immunotherapies and will hopefully contribute to further improved chances for cancer patients in the future.
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| 19. |
- Vanherberghen, Bruno, et al.
(författare)
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Classification of human natural killer cells based on migration behavior and cytotoxic response
- 2013
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 121:8, s. 1326-1334
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Tidskriftsartikel (refereegranskat)abstract
- Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas a minority of NK cells was responsible for a majority of the target cell deaths. These dynamic cytotoxicity data allowed categorization of NK cells into 5 distinct classes. A small but particularly active subclass of NK cells killed several target cells in a consecutive fashion. These "serial killers" delivered their lytic hits faster and induced faster target cell death than other NK cells. Fast, necrotic target cell death was correlated with the amount of perforin released by the NK cells. Our data are consistent with a model in which a small fraction of NK cells drives tumor elimination and inflammation.
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| 20. |
- 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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| 21. |
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| 22. |
- 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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| 23. |
- Wilkinson, Kai, et al.
(författare)
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Visualization of custom-tailored iron oxide nanoparticles chemistry, uptake, and toxicity
- 2012
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Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3364 .- 2040-3372. ; 4:23, s. 7383-7393
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Tidskriftsartikel (refereegranskat)abstract
- Nanoparticles of iron oxide generated by wearing of vehicles have been modelled with a tailored solution of size-uniform engineered magnetite particles produced by the Bradley reaction, a solvothermal metal-organic approach rendering hydrophilic particles. The latter does not bear any pronounced surface charge in analogy with that originating from anthropogenic sources in the environment. Physicochemical properties of the nanoparticles were thoroughly characterized by a wide range of methods, including XPD, TEM, SEM, DLS and spectroscopic techniques. The magnetite nanoparticles were found to be sensitive for transformation into maghemite under ambient conditions. This process was clearly revealed by Raman spectroscopy for high surface energy magnetite particles containing minor impurities of the hydromaghemite phase and was followed by quantitative measurements with EXAFS spectroscopy. In order to assess the toxicological effects of the produced nanoparticles in humans, with and without surface modification with ATP (a model of bio-corona formed in alveolar liquid), a pathway of potential uptake and clearance was modelled with a sequence of in vitro studies using A549 lung epithelial cells, lymphocyte 221-B cells, and 293T embryonal kidney cells, respectively. Raman microscopy unambiguously showed that magnetite nanoparticles are internalized within the A549 cells after 24 h co-incubation, and that the ATP ligand is retained on the nanoparticles throughout the uptake process. The toxicity of the nanoparticles was estimated using confocal fluorescence microscopy and indicated no principal difference for unmodified and modified particles, but revealed considerably different biochemical responses. The IL-8 cytokine response was found to be significantly lower for the magnetite nanoparticles compared to TiO2, while an enhancement of ROS was observed, which was further increased for the ATP-modified nanoparticles, implicating involvement of the ATP signalling pathway in the epithelium.
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| 24. |
- Wilkinson, Kai, et al.
(författare)
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Visualization of custom-tailored iron oxide nanoparticles chemistry, uptake, and toxicity
- 2012
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Ingår i: Nanoscale. - 2040-3364. ; 4:23, s. 7383-7393
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Tidskriftsartikel (refereegranskat)abstract
- Nanoparticles of iron oxide generated by wearing of vehicles have been modelled with a tailored solution of size-uniform engineered magnetite particles produced by the Bradley reaction, a solvothermal metal–organic approach rendering hydrophilic particles. The latter does not bear any pronounced surface charge in analogy with that originating from anthropogenic sources in the environment. Physicochemical properties of the nanoparticles were thoroughly characterized by a wide range of methods, including XPD, TEM, SEM, DLS and spectroscopic techniques. The magnetite nanoparticles were found to be sensitive for transformation into maghemite under ambient conditions. This process was clearly revealed by Raman spectroscopy for high surface energy magnetite particles containing minor impurities of the hydromaghemite phase and was followed by quantitative measurements with EXAFS spectroscopy. In order to assess the toxicological effects of the produced nanoparticles in humans, with and without surface modification with ATP (a model of bio-corona formed in alveolar liquid), a pathway of potential uptake and clearance was modelled with a sequence of in vitro studies using A549 lung epithelial cells, lymphocyte 221-B cells, and 293T embryonal kidney cells, respectively. Raman microscopy unambiguously showed that magnetite nanoparticles are internalized within the A549 cells after 24 h co-incubation, and that the ATP ligand is retained on the nanoparticles throughout the uptake process. The toxicity of the nanoparticles was estimated using confocal fluorescence microscopy and indicated no principal difference for unmodified and modified particles, but revealed considerably different biochemical responses. The IL-8 cytokine response was found to be significantly lower for the magnetite nanoparticles compared to TiO2, while an enhancement of ROS was observed, which was further increased for the ATP-modified nanoparticles, implicating involvement of the ATP signalling pathway in the epithelium.
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