| 1. |
- Haroun-Izquierdo, Alvaro, et al.
(författare)
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Adaptive single-KIR(+)NKG2C(+) NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia
- 2022
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Ingår i: Journal for ImmunoTherapy of Cancer. - : BMJ. - 2051-1426. ; 10:11, s. e005577-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Carannante, Valentina, et al.
(författare)
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Generation of tumor spheroids in microwells to study NK cell cytotoxicity, infiltration and phenotype
- 2023
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Ingår i: Methods in Cell Biology. - : Elsevier BV. - 0091-679X. ; , s. 195-208
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
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| 4. |
- Hammer, Quirin, et al.
(författare)
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Combined Genetic Ablation of CD54 and CD58 in CAR Engineered Cytotoxic Lymphocytes Effectively Averts Allogeneic Immune Cell Rejection
- 2022
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 140:Supplement 1, s. 1165-1166
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 5. |
- Hammer, Quirin, et al.
(författare)
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Genetic ablation of adhesion ligands averts rejection of allogeneic immune cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A significant barrier to the broad clinical implementation of allogeneic cell therapies is rejection of the allogeneic cells by the recipient immune system. Silencing of β-2-microglobulin (B2M) expression is a commonly employed strategy to evade T cell mediated rejection; however, the absence of B2M triggers missing-self responses by recipient natural killer (NK) cells. Here, we demonstrate that deletion of the adhesionligands CD54 and CD58 on allogeneic cells dampens recipient NK cell reactivityindependent of NK cell sub-population and inhibitory receptor expression. Additionally,genetic ablation of CD54 and CD58 in B2M-deficient allogeneic CAR T cells andinduced pluripotent stem cell-derived NK (iPSC-NK) cells reduces their susceptibility to rejection by recipient NK cells both in vitro and in vivo without affecting their anti-tumor effector potential. Thus, these data show that genetic ablation of adhesion ligands effectively mitigates rejection of allogeneic immune cells, enabling the generation of rejection-resistant allogeneic cell products.
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| 6. |
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| 7. |
- Prager, Isabel, et al.
(författare)
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NK cells switch from granzyme B to death receptor–mediated cytotoxicity during serial killing
- 2019
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 7:9, s. 2113-2127
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Sandström, Niklas, 1981-, et al.
(författare)
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Miniaturized and multiplexed high-content screening of drug and immune sensitivity in a multichambered microwell chip
- 2022
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Ingår i: CELL REPORTS METHODS. - : Elsevier BV. - 2667-2375. ; 2:7
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- van Ooijen, Hanna, et al.
(författare)
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Distinct mechanistic responses in serial-killing NK cells during natural and antibody-dependent cytotoxicity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Natural Killer (NK) cells are potent cytotoxic immune cells, yet the population exhibits a large phenotypical and functional heterogeneity. Here we investigated the mechanisms underlying NK cell serial killing during natural and antibody-dependent cellular cytotoxicity (ADCC). We leveraged on our microwell chip platform to relate cellular function with organelle content and intracellular protein expression, in individual NK cells. We observed that highly potent serial-killing NK cells more often deployed degranulation and induced necrosis, and that they were multifunctional, producing INF-γ and TNF-α. We probed for intrinsic differences between NK cells that performed serial killing compared to other NK cells, yet found no distinction in the migration dynamics, nor in the lysosomal and mitochondrial load of the cells. However, when measuring the calcium signaling of the cells in consecutive interactions, we revealed that serial-killing NK cells were able to maintain calcium signaling longer than cells that interrupted their killing sequence. By staining the cells at the end of the cytotoxicity assay, we found that NK cells that had stopped killing still showed clear intracellular expression of granzyme B-positive granules. Thus, it appears that decreased signaling rather than depletion of lytic cargo is the reason for NK cell exhaustion in our assay. Our results motivate exploring ways to enhance the signaling machinery in NK cells to achieve sustained functional responses.
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| 10. |
- van Ooijen, Hanna
(författare)
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Mechanisms regulating Natural Killer Cell Cytotoxicity
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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