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Olofsson, P. E., Brandt, L., Magnusson, K. E. G., Frisk, T., Jaldén, J. & Önfelt, B. (2019). A collagen-based microwell migration assay to study NK-target cell interactions. Scientific Reports, 9, Article ID 10672.
Open this publication in new window or tab >>A collagen-based microwell migration assay to study NK-target cell interactions
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 10672Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-255740 (URN)10.1038/s41598-019-46958-3 (DOI)000476718900058 ()31337806 (PubMedID)2-s2.0-85069667997 (Scopus ID)
Note

QC 20190812

Available from: 2019-08-12 Created: 2019-08-12 Last updated: 2019-08-12Bibliographically approved
Verron, Q., Guldevall, K., Brandt, L., Olofsson, P. E., Frisk, T. & Önfelt, B. (2017). Microchip screening for single cell assessment and isolation of serial killing NK cells. Paper presented at 44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN. Scandinavian Journal of Immunology, 86(4), 347-347
Open this publication in new window or tab >>Microchip screening for single cell assessment and isolation of serial killing NK cells
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2017 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 347-347Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
WILEY, 2017
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-215798 (URN)000411865200230 ()
Conference
44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN
Note

QC 20171018

Available from: 2017-10-18 Created: 2017-10-18 Last updated: 2017-10-18Bibliographically approved
Guldevall, K., Brandt, L., Forslund, E., Olofsson, K., Frisk, T. W., Olofsson, P. E., . . . Önfelt, B. (2016). Microchip screening Platform for single cell assessment of NK cell cytotoxicity. Frontiers in Immunology, 7, Article ID 119.
Open this publication in new window or tab >>Microchip screening Platform for single cell assessment of NK cell cytotoxicity
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2016 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 7, article id 119Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2016
Keywords
NK cells, cytotoxicity, single cell analysis, microchip, screening, microscopy, fluorescence, immune synapse
National Category
Immunology
Identifiers
urn:nbn:se:kth:diva-185604 (URN)10.3389/fimmu.2016.00119 (DOI)000373340600001 ()2-s2.0-84966702164 (Scopus ID)
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research Swedish Childhood Cancer FoundationSwedish Cancer Society
Note

QC 20160428

Available from: 2016-04-28 Created: 2016-04-25 Last updated: 2017-11-30Bibliographically approved
Forslund, E., Sohlberg, E., Enqvist, M., Olofsson, P. E., Malmberg, K.-J. & Önfelt, B. (2015). Microchip-Based Single-Cell Imaging Reveals That CD56(dim) CD57(-)KIR(-)NKG2A(+) NK Cells Have More Dynamic Migration Associated with Increased Target Cell Conjugation and Probability of Killing Compared to CD56(dim)CD57(-)KIR(-)NKG2A(-) NK Cells. Journal of Immunology, 195(7), 3374-3381
Open this publication in new window or tab >>Microchip-Based Single-Cell Imaging Reveals That CD56(dim) CD57(-)KIR(-)NKG2A(+) NK Cells Have More Dynamic Migration Associated with Increased Target Cell Conjugation and Probability of Killing Compared to CD56(dim)CD57(-)KIR(-)NKG2A(-) NK Cells
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2015 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 195, no 7, p. 3374-3381Article in journal (Refereed) Published
Abstract [en]

NK cells are functionally educated by self-MHC specific receptors, including the inhibitory killer cell Ig-like receptors (KIRs) and the lectin-like CD94/NKG2A heterodimer. Little is known about how NK cell education influences qualitative aspects of cytotoxicity such as migration behavior and efficacy of activation and killing at the single-cell level. In this study, we have compared the behavior of FACS-sorted CD56(dim)CD57(-)KIR(-)NKG2A(+) (NKG2A(+)) and CD56(dim)CD57(-)KIR(-)NKG2A(+) (lacking inhibitory receptors; IR-) human NK cells by quantifying migration, cytotoxicity, and contact dynamics using microchip-based live cell imaging. NKG2A(+) NK cells displayed a more dynamic migration behavior and made more contacts with target cells than IR-NK cells. NKG2A(+) NK cells also more frequently killed the target cells once a conjugate had been formed. NK cells with serial killing capacity were primarily found among NKG2A(+) NK cells. Conjugates involving IR- NK cells were generally more short-lived and IR- NK cells did not become activated to the same extent as NKG2A(+) NK cells when in contact with target cells, as evident by their reduced spreading response. In contrast, NKG2A(+) and IR- NK cells showed similar dynamics in terms of duration of conjugation periods and NK cell spreading response in conjugates that led to killing. Taken together, these observations suggest that the high killing capacity of NKG2A(+) NK cells is linked to processes regulating events in the recognition phase of NK-target cell contact rather than events after cytotoxicity has been triggered.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Immunology
Identifiers
urn:nbn:se:kth:diva-175487 (URN)10.4049/jimmunol.1500171 (DOI)000361741200043 ()26320254 (PubMedID)2-s2.0-84942475118 (Scopus ID)
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilSwedish Cancer SocietyScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20151028

Available from: 2015-10-28 Created: 2015-10-16 Last updated: 2017-12-01Bibliographically approved
Olofsson, P. E., Forslund, E., Vanherberghen, B., Chechet, K., Mickelin, O., Rivera Ahlin, A., . . . Önfelt, B. (2014). Distinct Migration and Contact Dynamics of Resting and IL-2-Activated Human Natural Killer Cells.. Frontiers in immunology, 5, 80
Open this publication in new window or tab >>Distinct Migration and Contact Dynamics of Resting and IL-2-Activated Human Natural Killer Cells.
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2014 (English)In: Frontiers in immunology, ISSN 1664-3224, Vol. 5, p. 80-Article in journal (Refereed) Published
Abstract [en]

Natural killer (NK) cells serve as one of the first lines of defense against viral infections and transformed cells. NK cell cytotoxicity is not dependent on antigen presentation by target cells, but is dependent on integration of activating and inhibitory signals triggered by receptor-ligand interactions formed at a tight intercellular contact between the NK and target cell, i.e., the immune synapse. We have studied the single-cell migration behavior and target-cell contact dynamics of resting and interleukin (IL)-2-activated human peripheral blood NK cells. Small populations of NK cells and target cells were confined in microwells and imaged by fluorescence microscopy for >8 h. Only the IL-2-activated population of NK cells showed efficient cytotoxicity against the human embryonic kidney 293T target cells. We found that although the average migration speeds were comparable, activated NK cells showed significantly more dynamic migration behavior, with more frequent transitions between periods of low and high motility. Resting NK cells formed fewer and weaker contacts with target cells, which manifested as shorter conjugation times and in many cases a complete lack of post-conjugation attachment to target cells. Activated NK cells were approximately twice as big as the resting cells, displayed a more migratory phenotype, and were more likely to employ "motile scanning" of the target-cell surface during conjugation. Taken together, our experiments quantify, at the single-cell level, how activation by IL-2 leads to altered NK cell cytotoxicity, migration behavior, and contact dynamics.

National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-146251 (URN)10.3389/fimmu.2014.00080 (DOI)000354057200001 ()24639676 (PubMedID)2-s2.0-84897939167 (Scopus ID)
Note

QC 20140611

Available from: 2014-06-11 Created: 2014-06-10 Last updated: 2017-01-09Bibliographically approved
Olofsson, P. (2014). Quantitative approaches to studying NK cell functional heterogeneity. (Licentiate dissertation). Stockholm: KTH Royal Institute of Technology
Open this publication in new window or tab >>Quantitative approaches to studying NK cell functional heterogeneity
2014 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

It is commonly stated that the cell is the smallest functional unit of life. By analogy, then, the immune cell is the smallest functional unit of the immune system. Natural killer (NK) cells are effector cells of the innate immune system that are responsible for mediating cellular cytotoxicity against virally infected or neoplastically transformed cells. Many phenotypically distinct subpopulations of NK cells have been discovered, usually by dividing cells on the basis of cell-surface markers. These subpopulations are typically described as related to activation or developmental status of the cells. However, how these distinct phenotypes correlate with behavior in e.g. NK–target interactions is less widely understood. There is therefore a need to study NK cell behavior down at the single-cell level. The aim of this thesis is to approach methods that quantitatively describe these single-cell-level behavioral differences of NK cells.

Using a newly developed single-cell imaging and screening assay, we trap small populations of NK and target cells inside microwells, where they can be imaged over extended periods of time. We have performed experiments on both resting and IL-2-activated NK cells and quantified their cytotoxic behavior. One major discovery was that a small population of NK cells mediate a majority of the cytotoxicity directed against target cells. A particularly cytotoxic group of cells, which we termed “serial killers”, displayed faster and more effective cytotoxicity.

Also, we identified differences between resting and activated NK cells in regard to their migration and contact dynamics. Activated NK cells were found to more readily adhere to targets cells than did NK cells freshly isolated from peripheral blood. Apart form migration and contact dynamics, we have also quantified killing behavior, where NK cells can be seen to exhibit a behavior we term multiple lytic hits on the basis of analyzing target cell fluorescence profiles.

We have quantified these heterogeneities and developed tools that can be used to further study and elucidate differences in the behavior of single immune cells. These methods, and automated single-cell analysis methods, will likely play a more important role in the study of immune responses in the future.

 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. p. xiii, 35
Series
TRITA-FYS, ISSN 0280-316X ; 2014:21
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-146281 (URN)978-91-7595-166-9 (ISBN)
Presentation
2014-05-27, sal Air, Gamma, Science for Life Laboratory, Science for Life Laboratory, Solna, 13:00 (English)
Opponent
Supervisors
Note

QC 20140611

Available from: 2014-06-11 Created: 2014-06-11 Last updated: 2014-06-11Bibliographically approved
Vanherberghen, B., Olofsson, P. E., Forslund, E., Sternberg-Simon, M., Khorshidi, M. A., Pacouret, S., . . . Önfelt, B. (2013). Classification of human natural killer cells based on migration behavior and cytotoxic response. Blood, 121(8), 1326-1334
Open this publication in new window or tab >>Classification of human natural killer cells based on migration behavior and cytotoxic response
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2013 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 121, no 8, p. 1326-1334Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
Apoptosis, Cell Communication, Cell Degranulation, Cell Movement, HEK293 Cells, Humans, Immunophenotyping, Killer Cells, Natural, Lymphocyte Activation, Microchip Analytical Procedures, Models, Biological, Necrosis, T-Lymphocytes, Cytotoxic
National Category
Hematology
Identifiers
urn:nbn:se:kth:diva-125773 (URN)10.1182/blood-2012-06-439851 (DOI)000321750000017 ()2-s2.0-84874447340 (Scopus ID)
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20130814

Available from: 2013-08-14 Created: 2013-08-13 Last updated: 2017-12-06Bibliographically approved
Forslund, E., Guldevall, K., Olofsson, P. E., Frisk, T., Christakou, A. E., Wiklund, M. & Önfelt, B. (2012). Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations. Frontiers in Immunology, 3(OCT), 300
Open this publication in new window or tab >>Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations
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2012 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 3, no OCT, p. 300-Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
Cell migration, Cytotoxicity, Live cell imaging, Microchip, NK cell, Single cell, Ultrasound
National Category
Other Physics Topics
Identifiers
urn:nbn:se:kth:diva-119183 (URN)10.3389/fimmu.2012.00300 (DOI)2-s2.0-84874217873 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20130311

Available from: 2013-03-11 Created: 2013-03-08 Last updated: 2017-12-06Bibliographically approved
Guldevall, K., Gustafsson, K., Forslund, E., Frisk, T., Manneberg, O., Olofsson, P. E., . . . Önfelt, B.Microchip screening platform for assessment of cytotoxic effector cells.
Open this publication in new window or tab >>Microchip screening platform for assessment of cytotoxic effector cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

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.

National Category
Immunology in the medical area
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-142469 (URN)
Funder
Swedish Foundation for Strategic Research Swedish Cancer SocietySwedish Research Council
Note

QS 2014

Available from: 2014-03-05 Created: 2014-03-05 Last updated: 2018-01-11Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-6019-8157

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