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| 2. |
- Brandt, Ludwig
(författare)
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NK Cell Cytotoxicity at the Single Cell Level
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Natural killer (NK) cells are innate immune cells with the ability to recognize and eliminate virally infected cells and cancer cells without prior sensitization. There is a functional heterogeneity between individual NK cells, where some NK cells are more efficient at killing cancer cells than others. Methods that allow studies of single NK cells are required to understand the functional differences and how they correlate with the activation and development status of the NK cell.This thesis focuses on the development and implementation of microchip- based imaging of NK cells, which is covered in five papers. Paper I presents a microchip screening platform for assessment of the cytotoxic potential of individual NK cells, by confining single NK cells together with target cells in microwells, followed by microscopy screening over extended time periods and automated image analysis. In paper II, the microchip platform was applied to test the ability of a novel trispecific killer engager (TriKE) to mediate an NK cell-dependent immune response. The process of NK cell education was studied in paper III and for that the image analysis methods for the microchip platform was further developed, in order to reveal new insight into how the education process affects the cytotoxic function of single NK cells. In paper IV, a previously developed microchip assay was extended to study NK cell migration and cytotoxicity in a more in vivo-like 3D collagen matrix. Paper V shows how NK cells can eliminate platelets in the presence of anti-platelet antibodies.In summary, this thesis covers the development and applications of time- lapse imaging using microwells for studying important NK cell functions in different settings. Understanding NK cell heterogeneity has the potential for improving e.g. cancer cell therapies.
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| 4. |
- 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
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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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| 6. |
- Oei, Vincent Yi Sheng, et al.
(författare)
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Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors
- 2018
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Ingår i: CANCER IMMUNOLOGY RESEARCH. - : AMER ASSOC CANCER RESEARCH. - 2326-6066 .- 2326-6074. ; 6:4, s. 467-480
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Tidskriftsartikel (refereegranskat)abstract
- Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19(+) targets, and maintained their intrinsic degranulation response toward CD19(-) K562 cells. The response of redirected NK-cell subsets against CD19(+) targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA-specific KIR(s), displayed superior ability to kill CD19(+), HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells.
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| 7. |
- Olofsson, Per E., et al.
(författare)
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A collagen-based microwell migration assay to study NK-target cell interactions
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Natural killer (NK) cell cytotoxicity in tissue is dependent on the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment. Traditional imaging studies of NK cell migration and cytotoxicity have utilized 2D surfaces, which do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. Here, we have combined a microwell assay that allows long-term imaging and tracking of small, well-defined populations of NK cells with an interstitial ECM-like matrix. The assay allows for long-term imaging of NK-target cell interactions within a confined 3D volume. We found marked differences in motility between individual cells with a small fraction of the cells moving slowly and being confined to a small volume within the matrix, while other cells moved more freely. A majority of NK cells also exhibited transient variation in their motility, alternating between periods of migration arrest and movement. The assay could be used as a complement to in vivo imaging to study human NK cell heterogeneity in migration and cytotoxicity.
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| 8. |
- Philippon, Camille, et al.
(författare)
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Allelic variation of KIR and HLA tunes the cytolytic payload and determines functional hierarchy of NK cell repertoires
- 2023
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Ingår i: Blood Advances. - : American Society of Hematology. - 2473-9529 .- 2473-9537. ; 7:16, s. 4492-4504
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Tidskriftsartikel (refereegranskat)abstract
- The functionality of natural killer (NK) cells is tuned during education and is associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation in killer cell immunoglobulin-like receptor (KIR) and HLA, which is known to influence the functional strength of NK cells, fine-tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but were stable over time in each individual and genetically determined by allelic variation in HLA class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of major histocompatibility complex-deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells, resulting in predictable hierarchies in global NK cell function.
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| 9. |
- 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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| 10. |
- 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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