The immune system provides defense against infectious agents such as viruses, bacteria and parasites. Besides eliminating extracellular agents, the immune system also constantly monitors our own cells for signs of transformation, including tumor development and virus infection. This process, called immune surveillance, is mediated in part by natural killer (NK) cells. NK cells sense transformation through the interaction of surface receptors with proteins on the surface of the diseased cell. The efficient binding of these receptors results in the formation of a tight contact between the two cells, called an immune synapse. If danger signals dominate in the synapse, the NK cell has the potential to deliver toxic compounds and to bind to specific death receptors at the target cell surface, resulting in the induction of target cell death. Apart from the ability to eliminate transformed cells, NK cells also have an immuno-regulatory function by directly killing other immune cells and by secreting pro- and anti-inflammatory cytokines.

Because of these roles, NK cells are of special interest in the growing field of cancer immunotherapy, where the function of immune cells is enhanced to defeat tumor cells. Clinical trials using NK cell-centered therapy have shown promising results against blood-borne cancer, yet progress has been limited against solid tumors. One possible explanation is related to the locally immuno-suppressive environment created by the solid tumor, for which improved research models are necessary. Besides, there is growing evidence of pronounced heterogeneity in the function of individual cells amidst the NK cell pool. Improving our understanding of NK cell biology thus requires advances in dedicated single-cell assays. For this purpose, our research group has previously developed miniaturized multi-well chips where individual cells can be confined and followed by microscopy over periods of several days. Using these microchips, a peculiar group of highly potent NK cells has been identified, which are able to kill several target cells in a row and contribute disproportionately to the overall cytotoxicity, and are therefore referred to as serial-killing NK cells.

The work presented in this thesis is focused on developing and applying microscopy-based single-cell assays to the study of NK cell functional heterogeneity, with a particular focus on the mechanistic aspects of cytotoxicity. In Paper I, we investigated the formation and outcome of immune synapses in single cells, using micro-patterning to create distinct spatial distributions of ligands. We observed that synapse formation was guided by the overall shape of the ligands while local signaling regulated the final steps of exocytosis. Paper II is dedicated to the study of the cytotoxic mechanisms used by individual NK cells and their regulation, in particular comparing serial-killing NK cells and moderate killers. Using dedicated fluorescent reporters, we identified a switch between two commonly used killing pathways, degranulation and death ligand engagement, and proposed a model for the underlying process. This topic was further detailed in Paper III, where the contribution of these cytotoxic mechanisms under additional antibody stimulation was studied. The investigation was conducted in a newly developed single-use plastic microchip, designed to enable the generation of multiple simultaneous two- and three-dimensional cell cultures while retaining high imaging performance. In Paper IV, we implemented single-cell retrieval from the silicon-glass microwells. We characterized the performance of our setup and demonstrated its potential at identifying and retrieving rare populations defined by functional readouts. 

Together, these studies further demonstrate the importance of single-cell analysis in the field of immunology. Besides advancing our understanding of NK cell biology, these developments may prove valuable in developing improved immunotherapies.

Over the last 50 years, cancer survival rates have steadily improved thanks to earlier detection and novel treatment regimens. For example, drugs that act on the immune system, so-called immunotherapy, have drastically increased the prospect of survival for patients suffering from some of the most aggressive cancer types including advanced malignant melanoma. Nevertheless, many patients still do not respond to neither traditional treatments or immunotherapy. Expanding the knowledge of immune cell function, and of how the immune system is dysregulated in cancer, will hence be fundamental for the development of more efficient treatment strategies.

Natural Killer (NK) cells, a type of cytotoxic innate immune cell, have been identified as a target for immunotherapy due to their capacity to recognize and destroy cancer cells. Tumor-infiltrating NK cells often display reduced functionality, and therapies targeting NK cells therefore aim at enhancing their anti-tumor activity. However, the responses of individual NK cells are highly heterogeneous, and although some cells efficiently destroy harmful targets, others do not.

Both functionality and phenotype are most efficiently studied using single-cell approaches, as these can resolve differences within heterogenous populations. In immunology, flow cytometry has been the golden standard for single-cell assessment, but this method has limited applicability when studying dynamic processes. For this purpose, imaging-based methods are a superior alternative, especially when combined with spatial confinement of single cells.

In this thesis, I describe the development and application of microscopy-based approaches for the study of single NK cell functional responses. Specifically, I have sought to increase our understanding of the mechanisms regulating NK cell cytotoxicity. In Paper 1, we developed a plastic microwell chip for the investigation of the function of NK cells at the single-cell level. The platform was used to examine how NK cell cytotoxicity is negatively affected by several factors present in the tumor microenvironment, including limited glucose and glutamine availability. In Paper 2, we explored the cytotoxic mechanisms deployed by individual NK cells during sequential killing. We showed that single NK cells switch from almost exclusively applying degranulation in early killing events, to using death ligand engagement for their final kill. In Paper 3, we further investigated the factors prohibiting NK cells from continued killing, during both natural ligand and antibody-mediated cytotoxicity. We discovered that most NK cells retain a large pool of granzyme B-positive lytic granules after they have ceased killing, but calcium signaling is maintained only in NK cells that are capable of sequential killing. In Paper 4, we explored ways of improving the therapeutic efficacy of in vitro-activated immune cells by avoiding their rejection by the host immune system. We showed that the combined deletion of CD54 and CD58 in grafted cells resulted in a reduced recognition by host NK cells, thereby improving graft survival.

In summary, the work presented in this thesis demonstrates the importance of single-cell methods for characterizing immune cell function. Such advancements are crucial for the continued development of immunotherapies and will hopefully contribute to further improved chances for cancer patients in the future.

De senaste 50 åren har chansen till överlevnad efter en cancerdiagnos ökat avsevärt tack vare tidigare diagnos och förbättrade behandlingsmetoder. Genom att rikta behandlingen mot immunförsvaret, så kallad immunterapi, har utsikterna för patienter som lider av särskilt elakartade cancertyper såsom avancerat malignt melanom förbättrats. Trots detta är det många patienter som varken svarar på traditionell behandling eller immunterapi. Ökad kunskap om mekanismerna som reglerar immuncellsmedierad tumörigenkänning och hur dessa påverkas under cancerutveckling är fundamentalt för utvecklingen av bättre behandlingsalternativ. 

Naturliga mördarceller, eller NK celler från engelskans Natural Killer cells, är en typ av cell som ingår i det medfödda immunförsvaret. Tack vare dess förmåga att känna igen och döda tumörceller utgör den en potentiell aktör vid immunterapi. NK celler hos cancerpatienter uppvisar ofta reducerad funktionalitet, och immunterapi inriktas därför mot att återställa eller förbättra cellernas cytotoxiska, celldödande potential. Enskilda NK celler har starkt varierande funktionalitet, vilket leder till att endast en fraktion av cellerna är effektiva.

För att studera enskilda cellers funktionalitet krävs en analysmetod som kan påvisa små skillnader i en stor population. Inom immunologi har flödescytometri varit golden standard för denna typ av analys, men metoden erbjuder inte möjligheten att studera dynamiska processer. För detta ändamål är mikroskopibaserade metoder överlägsna, särskilt då de kombineras med isolering av enskilda celler i droppar eller brunnar.

I denna avhandling beskriver jag utvecklingen och tillämpningen av mikroskopibaserade metoder för analys av enskilda NK-celler. Specifikt fokuserar jag på att öka vår förståelse för vilka mekanismer som reglerar NK-cellers cytotoxiska förmåga. I artikel 1 skildras hur vi utvecklade ett mikrobrunns-chip i plast. Genom att använda detta chip kunde vi visa att NK cellers cytotoxicitet påverkas negativt av faktorer i tumör-mikromiljön, som t.ex. låg tillgång till glukos och syre. I artikel 2 undersökte vi vilka cytotoxiska mekanismer NK-celler använder sig av under sekventiellt tumörcells-dödande. Vi kunde visa att cellerna byter från att tidigt under sekvensen använda degranulering till att slutligen i huvudsak engagera dödsligander. I artikel 3, byggde vi vidare på denna studie och undersökte vilka faktorer som hindrar NK-celler från att fortsätta dödandet. Vi fann att cellerna i slutet av den cytotoxiska sekvensen har granuler kvar, men att de tappar sin förmåga att upprätthålla kalciumsignalering. I artikel 4 studerade vi möjligheter till att förbättra livslängden för immunterapeutiska cellprodukter och visade att avstötningen av transplanterade immunceller kunde motverkas genom genetisk radering av CD54 och CD58 på cellytan.

Sammanfattningsvis åskådliggör arbetet i denna avhandling vikten av att analysera enskilda immunceller för att förstå det immunologiska svaret.  Enskild immuncellsanalys kommer att kunna bidra till fortsatt utveckling av immunterapier, vilket förhoppningsvis skall leda till ytterligare förbättrade chanser för cancerpatienter i framtiden.