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The impact of substrate stiffness on Natural Killer cell function
[Thesis]. Manchester, UK: The University of Manchester; 2019.
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Abstract
A diverse number of cell functions and behaviour are regulated by the mechanical properties of the surrounding tissue. Natural Killer (NK) cells kill infected or transformed cells either directly via the formation of lytic synapses at the cell-cell interface or indirectly via the secretion of pro-inflammatory cytokines. Diseased cells and inflamed tissue often present with altered mechanical properties and how this impacts NK cell function is currently unclear. Here, we set out to establish whether or not target cell stiffness impacts on NK cell cytotoxicity. To address this, human primary NK cells were placed on polyacrylamide substrates possessing a wide range of rigidities (0.5-120kPa) coated with antibodies against the activating receptor NKp30 and the integrin LFA-1. NK cells plated on activating substrates exhibited stronger activation with increasing substrate stiffness as demonstrated by enhanced cell spreading, and increased degranulation. Pro-inflammatory cytokine secretions showed quantitative changes with substrate stiffness with secretions highest on the stiffest substrates. To better model the 3D interaction between NK cells and target cells, we synthesised sodium alginate cell-sized microbeads, where bead stiffness was controlled to be low (10kPa), medium (40kPa), or high stiffness (230kPa). Beads were coated with antibodies against NKp30 and LFA-1 to create surrogate targets with specific ligands and controlled stiffness. Polarisation of the microtubule-organising centre (MTOC) and perforin towards the bead interface was dependent on target stiffness as both the MTOC and perforin failed to polarise towards soft targets. Consistent with this, the percentage of NK cells degranulating were higher when forming conjugates with stiffer targets. Thus, gel microbeads can be used to characterise the mechanosensitivity of immune synapses in 3D, and establish a role for target stiffness in NK cell activation. In addition, preliminary data indicated that NK cells migrating over fibronectin showed altered responses to substrate stiffness with enhanced motility, spreading and proliferation on stiffer surfaces. Taken together, our work demonstrates for the first time the mechanosensitive nature of the NK cell synapse and highlights the importance of mechanical signalling in a variety of key NK cell functions.