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Distinct Migration and Contact Dynamics of Resting and IL-2-Activated Human Natural Killer Cells.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0002-6019-8157
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
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2014 (English)In: Frontiers in immunology, ISSN 1664-3224, Vol. 5, 80- p.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.

Place, publisher, year, edition, pages
2014. Vol. 5, 80- p.
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:kth:diva-146251DOI: 10.3389/fimmu.2014.00080ISI: 000354057200001PubMedID: 24639676Scopus ID: 2-s2.0-84897939167OAI: oai:DiVA.org:kth-146251DiVA: diva2:723531
Note

QC 20140611

Available from: 2014-06-11 Created: 2014-06-10 Last updated: 2017-01-09Bibliographically approved
In thesis
1. Quantitative approaches to studying NK cell functional heterogeneity
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. xiii, 35 p.
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
2. Microscopy-based single-cell in vitro assays for NK cell function in 2-D and 3-D
Open this publication in new window or tab >>Microscopy-based single-cell in vitro assays for NK cell function in 2-D and 3-D
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. 88 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2017:02
National Category
Immunology
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-199571 (URN)978-91-7729-241-8 (ISBN)
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

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