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Microscopy-based single-cell in vitro assays for NK cell function in 2-D and 3-D
KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.ORCID iD: 0000-0002-6019-8157
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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. NK cell subsets are defined by their expression of certain cell-surface markers, and are usually related to activation and developmental status.

However, how distinct NK cell phenotypes correlate with behavior in NK-target interactions is less widely characterized. There is therefore a need to study NK cell behavior down at the single-cell level. One aim of this thesis is to approach methods that quantitatively describe these single-cell-level behavioral differences of NK cells.

Additionally, the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment is crucial for NK cell trafficking and immune surveillance. Traditional imaging studies of NK cell migration and cytotoxicity do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo.

Therefore, it is desirable to implement 3-D in vitro migration and killing assays that better mimic in vivo conditions. Another aim of this thesis is to develop a microwell-based assay for 3-D time-lapse imaging of NK cell migration and cytotoxicity.

Using a newly developed single-cell imaging and screening assay, we trap small populations of NK and target cells inside microwells, where they are imaged over extended periods of time. We have performed experiments on resting, IL-2-activated, educated, and non-educated NK cells and quantified their migration behavior and cytotoxicity. 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, termed serial killers, displayed faster and more effective cytotoxicity. Serial killers were more prevalent in IL-2-activated and educated NK cells, but were also present in a small fraction of resting and non-educated NK cells. IL-2-activated and educated NK cells displayed more dynamic migration behavior than resting and non-educated NK cells. Additionally, IL-2-activated and educated NK cells spent more time in NK–target cell conjugates and post-conjugation attachment than resting and non-educated NK cells.

To more closely approximate in vivo conditions, we have combined our microwell assay with an interstitial ECM-like matrix. The microwells allow for long-term imaging of NK–target cell interactions within a confined 3-D volume. NK cells were tracked and interactions with target cells were scored for duration and outcome. The developed microwell-based assay is suitable for 3-D time-lapse imaging of NK cell migration and cytotoxicity. As it allows for experiments with human cells, it could be used as a complement to in vivo imaging.

We have quantified NK cell behavioral heterogeneity and developed tools that can be used to further study and elucidate differences in the behavior of single immune cells. These tools advance current methods for single-cell analysis, which will likely play an even more important role in the study of immune responses in the future.

Abstract [sv]

NK-celler är effektorceller tillhörande det ospecifika immunförsvaret och har till uppgift att avdöda virusinfekterade och neoplastiska celler. Subpopulationer av NK-celler klassificeras på basis av uttryck av ytmolekyler och är vanligtvis relaterade till cellernas aktiverings- och utvecklingsstatus.

Hur dessa fenotypiskt distinkta subpopulationer korrelerar med beteende i NK–målcellinteraktioner är inte lika välstuderat. Det finns därför ett behov att studera NK-cellbeteende ner på encellsnivå. Ett mål med denna avhandling är att närma sig metoder som kvantitativt beskriver dessa skillnader i NK-cellbeteende på encellsnivå.

NK-cellers förmåga att migrera genom extracellulär matris är avgörande för deras celltrafik och immunövervakning. I traditionella avbildningsstudier av NK-cellers migration och cytotoxicitet återskapas inte de strukturella och mekaniska faktorer som formar NK-cellmigration in vivo.

Det är därför önskvärt att implementera migrationsassays i 3-D som bättre efterliknar in vivo-situationer. Ett annat mål med denna avhandling är att utveckla en mikrobrunnsbaserad assay för 3-D-avbildning av NK-cellmigration och -cytotoxicitet.

Genom att använda en nyligen utvecklad plattform för encellsavbildning och -screening fångar vi små populationer av NK- och målceller inuti mikrobrunnar, där de kan avbildas under längre tider. Vi har genomfört experiment på vilande och IL-2-aktiverade NK-celler, samt undersökt NK-cellutbildning, och kvantifierat dessa cellers migration och cytotoxiska beteende. En huvudsaklig upptäckt var att en liten population av de studerade NK-cellerna avdödade en majoritet av målcellerna. En särskilt cytotoxisk grupp celler, som benämnes seriemördare, uppvisade en snabbare och mer effektiv cytotoxicitet. Seriemördare var mer vanligt förekommande hos IL-2-aktiverade och utbildade NK-celler än hos vilande och icke-utbildade NK-celler. IL-2-aktiverade och utbildade NK-celler uppvisade mer dynamiskt migrationsbeteende än vilande och icke-utbildade NK-celler. Dessutom tillbringade IL-2-aktiverade och utbildade NK-celler en länge tid i målcellskonjugat och var i kontakt med målceller längre efter konjugering än vilande och icke-utbildade NK-celler.

För att närmare återskapa in vivo-tillstånd har vi kombinerat vår mikrobrunnsassay med en matris som liknar interstitiell extracellulär matris. Mikrobrunnarna möjliggör långtidsavbildning av NK–målcellinteraktioner inom en avgränsad volym. NK-cellerna spårades och längden och utfallet av målcellinteraktioner utvärderades. Den utvecklade mikrobrunnsassayen är lämplig för 3-D-avbildning av NK-cellmigration och -cytotoxicitet. Eftersom den tillåter experiment med humana celler kan den komplettera avbildning in vivo.

Vi har kvantifierat funktionell NK-cellheterogenitet och utvecklat verktyg som kan användas för att ytterligare studera och bringa klarhet i hur enskilda immuncellers beteende skiljer sig åt. Dessa verktyg är en vidareutveckling av nuvarande metoder för encellsanalys, som sannolikt kommer att spela en större roll i studiet av immunsvar i framtiden.

Abstract [zh]

自然杀伤细胞是先天免疫系统自带的效应细胞,主要通过调解其细胞毒性对抗病毒感染和细胞瘤变。自然杀伤细胞的亚型主要通过其表面抗原性质来定义并通常与一些激活和进展状态相联系。然而,关于自然杀伤细胞表型与其目标反应之间的相互联系的研究依然比较匮乏。因此,在单细胞层面对自然杀伤细胞表现的研究是十分必要的。

本论文的研究目的之一就是寻找方法来定量分析单细胞层水平NK细胞的行为差异。

此外,自然杀伤细胞在细胞外基质微环境中的迁移对自然杀伤细胞的移动和免疫监督非常重要。

关于自然杀伤细胞迁移和细胞毒性的传统成像研究并不能合理地呈现触发此细胞在体内迁移的形变和应变响应过程。因此,关于细胞迁移和细胞杀伤的体外三维研究对探索NK细胞的体内反应机制尤为重要。

本论文的另一个目的就是构建基于微孔试验来研究NK细胞迁移和细胞毒性随时间在三维空间中随时间的变化。

通过新型的单细胞成像和筛选方法,我们将少量NK细胞和靶细胞放入微孔内,同时进行长期的图像观察。 我们实验观察并测定了不同NK细胞的迁移和细胞毒性,包括静止型,IL-2 激活型,诱导型和非诱导型NK细胞。

一个重要发现是少量NK细胞实际上介导了其对靶细胞的主要细胞毒性。

一个具有特别细胞毒性的群体,称为连续杀伤细胞/持续杀伤细胞,表现出了更快更有效的细胞毒性。连续杀伤细胞在IL-2 激活型,诱导型细胞中出现得更多,但是在静止型和非诱导型细胞中也少量释放。前两者比后两者表现出了更活跃的迁移性能,但需要较长的结合时间。 

为了更接近在体状态,我们把基于微孔的实验与细胞外基质类似结构结合来研究NK细胞的活动。微孔有效地把NK细胞控制在一个三维小空间内,以便长时间观察NK细胞与目标细胞的反应。NK细胞可以被一直追踪并进一步测定了其与目标细胞的反应时间和反应结果。这种基于微孔的测试对研究NK细胞在三维空间内随时间的迁移和细胞毒性的图像研究非常有效。

它也适应于人类细胞的研究,可以为体内细胞成像研究提供良好辅助平台。

综上,本论文研究中,我们量化分析了NK细胞行为的异质性,并开发了实验方法可用于进一步研究和阐明不同单一免疫细胞的行为的方法。 

这些实验手段进一步提升了单细胞研究分析能力,并且未来将在免疫响应研究进一步起到更加重要的作用。

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , p. 88
Series
TRITA-FYS, ISSN 0280-316X ; 2017:02
National Category
Immunology
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-199571ISBN: 978-91-7729-241-8 (print)OAI: oai:DiVA.org:kth-199571DiVA, id: diva2:1062979
Public defence
2017-01-13, Air/Fire Conference Rooms, Tomtebodavägen 23A, Solna, 09:15 (English)
Opponent
Supervisors
Note

QC 20170110

Available from: 2017-01-10 Created: 2017-01-09 Last updated: 2017-01-10Bibliographically approved
List of papers
1. Classification of human natural killer cells based on migration behavior and cytotoxic response
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
2. Distinct Migration and Contact Dynamics of Resting and IL-2-Activated Human Natural Killer Cells.
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
3. 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
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
4. Microchip screening Platform for single cell assessment of NK cell cytotoxicity
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
5. A collagen-based microwell migration assay to study NK—target cell interactions
Open this publication in new window or tab >>A collagen-based microwell migration assay to study NK—target cell interactions
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Natural killer (NK) cell cytotoxicity is highly 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 2-D surfaces, which do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. In addition, current in vivo imaging does not allow for the accurate long-term single-cell imaging required to dissect the functional heterogeneity of NK cell populations, and importantly, it does not allow studies of human cells. Therefore, it is desirable to implement in vitro migration and killing assays that better mimic in vivo conditions.

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 to more closely approximate in vivo conditions. The microwells, which are loaded with a gel mixture containing NK and target cells, allows for long-term imaging of NK–target cell interactions within a confined 3-D volume. The microwells were optically sectioned by confocal fluorescence microscopy once every 2 min for 12 h. NK cells were tracked by the Baxter Algorithms to assess motility parameters and interactions with target cells were manually scored for duration and outcome.

We found marked differences in motility between individual cells with a significant fraction of the cells moving slowly and being confined to a small area within the matrix, while other cells moved more freely, probably reflecting local variations in the matrix structure and inherent difference in motility between individual cells. A majority of NK cells also exhibited transient variation in their mobility alternating between periods of migration arrest and random movement. NK cells that alternated between different modes of migration switched on average once every 3 h.

NK cells made fewer and shorter contacts with target cells than in comparable 2-D assays. The difference was particularly pronounced for the process of post-conjugation attachment when NK and target cells separate. The timing of this process is likely influenced by a biomechanical component only present in 3-D environments where the cells are offered multiple anchor points with the matrix that can be used to generate the forces needed to pull apart.

The developed microwell-based assay is suitable for 3-D time-lapse imaging of NK cells migration and cytotoxicity. As it allows for experiments with human cells, it could be used as a complement to in vivo imaging to study the influence of e.g. education and cytokine activation on NK cell heterogeneity in migration and cytotoxicity.

National Category
Immunology
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-199454 (URN)
Note

QCR 20170109

Available from: 2017-01-09 Created: 2017-01-09 Last updated: 2017-01-09Bibliographically approved

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