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- 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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- Kamali-Zare, Padideh, 1980-, et al.
(författare)
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Diffusion limited space contributes to K+ siphoning by regulation of K+ and water homeostasis in astrocytes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Diffusion Limited Space (DLS) is defined as a region where diffusion is limited by the geometry. Two examples of DLS in the brain are the neuronal synapse, and the narrow region between astrocyte endfeet and blood capillaries. In a series of geometrical models we show that DLS plays a role in regulation of water and K+ homeostasis in the brain by an indirect functional coupling of aquaporins (AQPs) and inward rectifying K+ (Kir) channels in a membrane microdomain. 1. Simulations in geometrical models of a synapse region show that following a step increase in synaptic [K+], both K+ and water are taken up by astrocytes via AQPs and Kir channels lining the synapse. This uptake creates a transient depletion of water in the synapse region that, enhanced by the DLS, facilitates K+ uptake and an efficient clearance of excess K+ from the synapse. 2. Simulations in a geometrical model of astrocytes show that the DLS formed between astrocyte endfeet and blood capillaries, facilitate the siphoning of accumulated K+ into the extracellular space facing the blood capillaries. The DLS geometry creates an efficient coupling between AQPs and Kir channels. 3. Furthermore, the models show that a local coupling between water and K+ transport is important for the maintenance of membrane potential and the net K+ spatial buffering capacity in the astrocytes. 4. In the full geometrical model of K+ spatial buffering we show that the geometry of the extracellular space both in the synapse region and in the endfeet is an essential component for the cell volume regulation. Our results suggest that for regulation of K+ and water homeostasis in astrocytes, not only the classical aspects of functional couplings between proteins, but also the geometry of the cell and the microdomains are important. Further, our results suggest a central role for AQPs in the astrocyte endfeet and identify their contribution to K+ siphoning.
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- Olofsson, Per, et al.
(författare)
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A collagen-based microwell migration assay to study NK—target cell interactions
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Natural killer (NK) cell cytotoxicity is highly dependent on the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment. Traditional imaging studies of NK cell migration and cytotoxicity have utilized 2-D surfaces, which do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. In addition, current in vivo imaging does not allow for the accurate long-term single-cell imaging required to dissect the functional heterogeneity of NK cell populations, and importantly, it does not allow studies of human cells. Therefore, it is desirable to implement in vitro migration and killing assays that better mimic in vivo conditions.We have combined a microwell assay that allows long-term imaging and tracking of small, well-defined populations of NK cells with an interstitial ECM-like matrix to more closely approximate in vivo conditions. The microwells, which are loaded with a gel mixture containing NK and target cells, allows for long-term imaging of NK–target cell interactions within a confined 3-D volume. The microwells were optically sectioned by confocal fluorescence microscopy once every 2 min for 12 h. NK cells were tracked by the Baxter Algorithms to assess motility parameters and interactions with target cells were manually scored for duration and outcome.We found marked differences in motility between individual cells with a significant fraction of the cells moving slowly and being confined to a small area within the matrix, while other cells moved more freely, probably reflecting local variations in the matrix structure and inherent difference in motility between individual cells. A majority of NK cells also exhibited transient variation in their mobility alternating between periods of migration arrest and random movement. NK cells that alternated between different modes of migration switched on average once every 3 h.NK cells made fewer and shorter contacts with target cells than in comparable 2-D assays. The difference was particularly pronounced for the process of post-conjugation attachment when NK and target cells separate. The timing of this process is likely influenced by a biomechanical component only present in 3-D environments where the cells are offered multiple anchor points with the matrix that can be used to generate the forces needed to pull apart.The developed microwell-based assay is suitable for 3-D time-lapse imaging of NK cells migration and cytotoxicity. As it allows for experiments with human cells, it could be used as a complement to in vivo imaging to study the influence of e.g. education and cytokine activation on NK cell heterogeneity in migration and cytotoxicity.
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- 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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- van Ooijen, Hanna, et al.
(författare)
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Screening and high-resolution imaging of dynamic single-cell responses in 2D and 3D using a novel disposable microwell chip
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Single-cell assays are developed as novel tools for the identification and characterization of individual cell responses. Some methods provide snapshots of the phenotype of the cell, such as flow cytometry and single-cell RNA sequencing, whereas others, almost exclusively microscopy-based, can be used for longitudinal studies of individual cells. In the present study, we developed 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 cell cultures. We confirm its 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 demonstrate the applicability of the system for single-cell analysis by correlating the dynamic cytotoxic response of single immune cells 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 NK cell-induced killing. Following the functional screening, we perform high-resolution 3D immunofluorescent imaging of infiltrating NK cells within the spheroid volumes.
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