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Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments is critical for their functions under physiological conditions. Recent studies have shown that NK cell activation is regulated by substrate stiffness. However, the role of mechanosensing in regulating NK cell killing efficiency in physiologically relevant scenarios is poorly understood. In this study, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK cell killing efficiency in 3D is impaired against softened tumor cells, while it is enhanced against stiffened tumor cells. Notably, the durations required for NK cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel in NK cells. Perturbation of PIEZO1 by GsMTx4 abolishes stiffness-dependent NK cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK cell killing efficiency under physiological conditions and the influence of environmental physical cues on NK cell functions.
Engineered antigen-presenting hydrogels: model platforms for studies of T cell mechanotransduction
(2020)
T cells apply forces and eventually sense and then respond to the mechanical properties of their surroundings, including those of antigen presenting cells (APC) when they form the immunological synapse (IS). The identification of the mechanosensitive receptors and time scales at which they sense and actuate is experimentally difficult at the natural cell-cell interface. Inspired by the tools used in cell-matrix mechanobiology, this thesis presents synthetic, hydrogel-based models of APCs to study T cell mechanotransduction, focusing on the early T cell activation. Polyacrylamide (PAAm) hydrogels (1-50 kPa) were micropatterned with streptavidin and APC ligands (antibody against CD3 co-receptor (anti-CD3) and intercellular cell adhesion molecule-1 (ICAM-1)) at controlled ligand density and in geometries with defined dimensions. The anti-CD3 patterned hydrogels were used to study the interplay between hydrogel stiffness and CD3-mediated early T cell activation markers. In the last chapter, the regulatory role of ICAM-1 coupled to anti-CD3 and hydrogel stiffness in early T cell activation was studied on hydrogels with patterned anti-CD3 microdots surrounded by a background of ICAM-1. The results contribute to the understanding of the factors involved in T cell mechanotransduction, providing useful information for the future design of immunomodulatory materials.
Progress in our understanding of mechanotransduction events requires noninvasive methods for the manipulation of forces at molecular scale in physiological environments. Inspired by cellular mechanisms for force application (i.e. motor proteins pulling on cytoskeletal fibers), we present a unique molecular machine that can apply forces at cell-matrix and cell-cell junctions using light as an energy source. The key actuator is a light-driven rotatory molecular motor linked to polymer chains, which is intercalated between a membrane receptor and an engineered biointerface. The light-driven actuation of the molecular motor is converted in mechanical twisting of the entangled polymer chains, which will in turn effectively “pull” on engaged cell membrane receptors (e.g., integrins, T cell receptors) within the illuminated area. Applied forces have physiologically-relevant magnitude and occur at time scales within the relevant ranges for mechanotransduction at cell-friendly exposure conditions, as demonstrated in force-dependent focal adhesion maturation and T cell activation experiments. Our results reveal the potential of nanomotors for the manipulation of living cells at the molecular scale and demonstrate a functionality which at the moment cannot be achieved by other technologies for force application.
Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments are critical for their functions under physiological conditions. However, the role of mechanosensing in regulating NK-cell killing efficiency in physiologically relevant scenarios is poorly understood. Here, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK-cell killing efficiency in 3D is impaired against softened tumor cells, whereas it is enhanced against stiffened tumor cells. Notably, the durations required for NK-cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel among the examined candidates in NK cells. Perturbation of PIEZO1 abolishes stiffness-dependent NK-cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK-cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK-cell killing efficiency under 3D physiological conditions and the influence of environmental physical cues on NK-cell functions.
Effective T cell responses against tumor cells require diverse effector functions including polarization towards tumor cells to form immunological synapses and nuclear factor of activated T-cells (NFAT)-dependent gene transcription. While the role of tumor cell softening has been associated with malignancy, stemness, and metastasis, potentially contributing to immune evasion, its impact on cellular processes in T cells is not well understood. Here, we show that both T cell polarization and NFAT nuclear translocation are modulated by target stiffness in a Ca2+ dependent manner. Using both anti-CD3 antibody-functionalized substrates with varying stiffness as surrogates for target cells or softened tumor cells, we found that both, reorientation of microtubule organizing center (MTOC) towards the tumor cells, a hallmark for T cell polarization, and NFAT translocation were impaired on softer hydrogels or following contact with softer cancer cells. The amplitudes of intracellular Ca2+ signals were dependent on stiffness, and removal of extracellular Ca2+ inhibited stiffness-dependent T cell responsiveness. While stiffness-dependent Ca2+ signaling was crucial for both, T cell polarization and NFAT translocation, Ca2+ influx through Piezo1, a mechanosensitive ion channel, mediated stiffness-dependent MTOC reorientation but not NFAT translocation. In contrast, Ca2+ influx through store-operated Orai channels mediated NFAT translocation but not MTOC reorientation. Our results demonstrate that tumor cell stiffness directly influences T cell functionality through distinct Ca2+ influx pathways, revealing cell softening as an essential mechanism employed by malignant cells to evade immune surveillance.
