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Classification of human natural killer cells based on migration behavior and cytotoxic response
KTH, School of Engineering Sciences (SCI), Applied Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-6019-8157
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2013 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 121, no 8, 1326-1334 p.Article 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.

Place, publisher, year, edition, pages
2013. Vol. 121, no 8, 1326-1334 p.
Keyword [en]
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: urn:nbn:se:kth:diva-125773DOI: 10.1182/blood-2012-06-439851ISI: 000321750000017Scopus ID: 2-s2.0-84874447340OAI: oai:DiVA.org:kth-125773DiVA: diva2:640782
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
In thesis
1. Live Single Cell Imaging and Analysis Using Microfluidic Devices
Open this publication in new window or tab >>Live Single Cell Imaging and Analysis Using Microfluidic Devices
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Today many cell biological techniques study large cell populations where an average estimate of individual cells’ behavior is observed. On the other hand, single cell analysis is required for studying functional heterogeneities between cells within populations. This thesis presents work that combines the use of microfluidic devices, optical microscopy and automated image analysis to design various cell biological assays with single cell resolution including cell proliferation, clonal expansion, cell migration, cell-cell interaction and cell viability tracking. In fact, automated high throughput single cell techniques enable new studies in cell biology which are not possible with conventional techniques.

In order to automatically track dynamic behavior of single cells, we developed a microwell based device as well as a droplet microfluidic platform. These high throughput microfluidic assays allow automated time-lapse imaging of encapsulated single cells in micro droplets or confined cells inside microwells. Algorithms for automatic quantification of cells in individual microwells and micro droplets are developed and used for the analysis of cell viability and clonal expansion. The automatic counting protocols include several image analysis steps, e.g. segmentation, feature extraction and classification. The automatic quantification results were evaluated by comparing with manual counting and revealed a high success rate. In combination these automatic cell counting protocols and our microfluidic platforms can provide statistical information to better understand behavior of cells at the individual level under various conditions or treatments in vitro exemplified by the analysis of function and regulation of immune cells. Thus, together these tools can be used for developing new cellular imaging assays with resolution at the single cell level.

To automatically characterize transient migration behavior of natural killer (NK) cells compartmentalized in microwells, we developed a method for single cell tracking. Time-lapse imaging showed that the NK cells often exhibited periods of high motility, interrupted with periods of slow migration or complete arrest. These transient migration arrest periods (TMAPs) often overlapped with periods of conjugations between NK cells and target cells. Such conjugation periods sometimes led to cell-mediated killing of target cells. Analysis of cytotoxic response of NK cells revealed that a small sub-class of NK cells called serial killers was able to kill several target cells. In order to determine a starting time point for cell-cell interaction, a novel technique based on ultrasound was developed to aggregate NK and target cells into the center of the microwells. Therefore, these assays can be used to automatically and rapidly assess functional and migration behavior of cells to detect differences between health and disease or the influence of drugs.

The work presented in this thesis gives good examples of how microfluidic devices combined with automated imaging and image analysis can be helpful to address cell biological questions where single cell resolution is necessary. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. vi, 53 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2013:14
Keyword
Single cell analysis, time-lapse fluorescence imaging, automated image analysis, microwell, droplet microfluidics, NK cells, single cell tracking, migration behavior analysis, cell-cell interaction, optical microscopy, image analysis, image processing, microfluidics, immune cells, tracking, counting, morphology analysis
National Category
Other Medical Biotechnology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-129278 (URN)978-91-7501-846-1 (ISBN)
Public defence
2013-10-18, Gardaulan, Smittskyddsinstitutet, Nobels väg 18, Karolinska Institutet, Solna, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 70784
Note

QC 20130927

Available from: 2013-09-26 Created: 2013-09-25 Last updated: 2014-02-28Bibliographically approved
2. Single Cell Investigations of the Functional Heterogeneity Within Immune Cell Populations: a Microchip-based Study
Open this publication in new window or tab >>Single Cell Investigations of the Functional Heterogeneity Within Immune Cell Populations: a Microchip-based Study
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. viii, 61 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2014:08
National Category
Immunology in the medical area Immunology
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-142472 (URN)978-91-7595-028-0 (ISBN)
Public defence
2014-03-25, Air and Fire, Science for Life Laboratories, Tomtebodavägen 23A, 17165, Solna, 09:30 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Foundation for Strategic Research
Note

QC 20140306

Available from: 2014-03-06 Created: 2014-03-05 Last updated: 2014-03-06Bibliographically approved
3. 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
4. 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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