We present an easy-to-use, disposable, thermoplastic microwell chip designed to support screening and high-resolution imaging of single-cell behavior in two- and three-dimensional (2D and 3D) cell cultures. We show that the chip has excellent optical properties and provide simple protocols for efficient long-term cell culture of suspension and adherent cells, the latter grown either as monolayers or as hundreds of single, uniformly sized spheroids. We then demonstrate the applicability of the system for single-cell analysis by correlating the dynamic cytotoxic response of single immune cells grown under different metabolic conditions to their intracellular cytolytic load at the end of the assay. Additionally, we illustrate highly multiplex cytotoxicity screening of tumor spheroids in the chip, comparing the effect of environment cues characteristic of the tumor microenvironment on natural killer (NK)-cell-induced killing. Following the functional screening, we perform high-resolution 3D immunofluorescent imaging of infiltrating NK cells within the spheroid volumes.

Allogeneic cellular immunotherapies hold promise for broad clinical implementation but face limitations due to potential rejection of donor cells by the host immune system. Silencing of beta-2 microglobulin (B2M) B2M ) expression is commonly employed to evade T cell-mediated rejection by the host, although the absence of B2M is expected to trigger missing-self responses by host natural killer (NK) cells. Here, we demonstrate that genetic deletion of the adhesion ligands CD54 and CD58 in B2M-deficient chimeric antigen receptor (CAR) T cells and multi-edited induced pluripotent stem cell (iPSC)-derived CAR NK cells reduces their susceptibility to rejection by host NK cells in vitro and in vivo. . The absence of adhesion ligands limits rejection in a unidirectional manner in B2M-deficient and B2M-sufficient settings without affecting the antitumor functionality of the engineered donor cells. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection by host immune cells, facilitating the implementation of universal immunotherapy.

The development of new immunotherapeutic drugs and combinatorial strategies requires the implementation of novel methods to test their efficacy in vitro. Here, we present a series of miniaturized in vitro assays to assess immune cell cytotoxic activity, infiltration, and phenotype in renal carcinoma spheroids with the use of a recently developed multichambered microwell chip. We provide protocols for tumor spheroid formation, NK cell culture, fluorescence labelling and imaging of live or fixed cells directly in the chip together with data analysis.

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.

BackgroundNatural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)(+)NKG2C(+) adaptive NK cells to maximize missing-self reactivity.MethodsWe developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML.ResultsADAPT-NK cells were >90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45(dim) blast subtypes.ConclusionsThese preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.

Allogeneic cell therapies hold promise to be cost effective with scaled manufacturing for multi-dosing and on-demand off-the-shelf availability. A critical consideration for allogeneic cell products is their ability to persist, maintain function and avoid rejection by the patient's immune system. Genetic knockout (KO) of beta-2-microglobulin (B2M) leads to complete loss of cell-surface human leukocyte antigen (HLA) class I expression and efficiently abrogates CD8+ T-cell reactivity. However, loss of HLA class I triggers NK cell-mediated missing-self recognition and manipulation of B2M must therefore be combined with other immune-modulating strategies to limit recipient NK cell reactivity.We hypothesized that rejection by the patient's immune system can be diminished in primary CAR T cells, iPSC-derived T (iT) and NK (iNK) cells by reverse-engineering common tumor escape mechanisms. The adhesion molecules CD54 and CD58 are both present at the target cell side of the immune synapse, and loss of either of these molecules have previously been reported to elicit immune escape. Here, we show that the combined deletion of CD54 and CD58 in allogeneic immune effector cells makes them resistant to rejection by recipient immune cells through unidirectional reduced synapse formation (Figure 1A).HLA class I down-regulation by B2M silencing in primary T and NK cells triggered potent cytotoxicity by resting allogeneic NK cells. This response was mostly driven by educated NK cells expressing either NKG2A or killer cell immunoglobulin-like receptors (KIR) binding to HLA-E and HLA-C, respectively. However, over-expression of HLA-E or single HLA-C ligands in a K562 screening model only shut down the specific response of the NK cell subset carrying the cognate inhibitory receptor, resulting in only partial resistance to NK cells at the bulk level. Notably, the introduction of HLA-E was particularly detrimental in donors with expanded NKG2C+ NK cell subsets, due to its stimulatory effect through the activating NKG2C receptor. In contrast, combined deletion of CD54 and CD58 in target cells uniquely decreased the response of all tested NK cell subsets and showed universal reduction across NK cell populations from 18 healthy donors (Figure 1B). To delineate the mechanisms behind the increased resistance of target cells carrying these edits, we studied NK cell-target cell interactions at the single cell level by confocal microscopy in microchips. Allogeneic NK cells formed fewer conjugates and failed to form productive immune synapses with CD54-/-CD58-/- target cells, supporting the notion that they are more resistant to NK-cell mediated killing by unidirectional altered adhesion.We next introduced these edits in primary B2M-/- T cells engineered to express a second generation CAR19 from the TRAC locus. Corroborating the K562 screen, CD54-/-CD58-/-B2M-/- CAR-T cells had a selective survival advantage over B2M-/- CAR T cells and HLA-E-over-expressing B2M-/- CAR T cells in conventional mixed lymphocyte reaction (MLR) assays in vitro. Furthermore, we established an in vivo model to probe the effect of different genetic edits on the persistence of allogeneic cell therapy products. To this end, a mixed population of B2M-/- CAR T cells additionally bearing either CD54 and/or CD58 KO, HLA-E over-expression, or no further edits were infused into mice harboring allogeneic healthy donor PBMC. We found that CD54-/-CD58-/-B2M-/- CAR T cells had significantly better in vivo persistence compared to both B2M-/- CAR T cells and HLA-E+B2M-/- CAR T cells in the presence of PBMC from healthy donors (Figure 1B).Although multiplexed editing is feasible in primary CAR T cells, the iPSC platform has an unmatched capacity for homogenously introducing multiple immune-evasion strategies for off-the-shelf cell therapy. Similar to primary CAR T cells, multiplexed edited CD54-/-CD58-/-B2M-/-CIITA-/- iNK cells showed normal growth kinetics and were resistant to rejection by activated allogeneic NK cells in MLR assays.Together, these data demonstrate that reverse-engineering of common tumor escape mechanisms, which render target cells less susceptible to immune synapse formation, is an effective strategy to avert immune rejection of allogeneic CAR T and iPSC-derived CAR NK cells.

Here, we present a methodology based on multiplexed fluorescence screening of two-or three-dimensional cell cultures in a newly designed multichambered microwell chip, allowing direct assessment of drug or im-mune cell cytotoxic efficacy. We establish a framework for cell culture, formation of tumor spheroids, fluores-cence labeling, and imaging of fixed or live cells at various magnifications directly in the chip together with data analysis and interpretation. The methodology is demonstrated by drug cytotoxicity screening using ovarian and non-small cell lung cancer cells and by cellular cytotoxicity screening targeting tumor spheroids of renal carcinoma and ovarian carcinoma with natural killer cells from healthy donors. The miniaturized format allowing long-term cell culture, efficient screening, and high-quality imaging of small sample volumes makes this methodology promising for individualized cytotoxicity tests for precision medicine.

NK cells eliminate virus-infected and tumor cells by releasing cytotoxic granules containing granzyme B (GrzB) or by engaging death receptors that initiate caspase cascades. The orchestrated interplay between both cell death pathways remains poorly defined. Here we simultaneously measure the activities of GrzB and caspase-8 in tumor cells upon contact with human NK cells. We observed that NK cells switch from inducing a fast GrzB-mediated cell death in their first killing events to a slow death receptor–mediated killing during subsequent tumor cell encounters. Target cell contact reduced intracellular GrzB and perforin and increased surface-CD95L in NK cells over time, showing how the switch in cytotoxicity pathways is controlled. Without perforin, NK cells were unable to perform GrzB-mediated serial killing and only killed once via death receptors. In contrast, the absence of CD95 on tumor targets did not impair GrzB-mediated serial killing. This demonstrates that GrzB and death receptor–mediated cytotoxicity are differentially regulated during NK cell serial killing.