The impact of target stiffness and tissue localisation on NK cell cytotoxicity

Abstract

Natural Killer (NK) cells play a fundamental role in immunosurveillance by identifying and killing virally infected or transformed cells. They do this through the lytic immune synapse, which forms at the interface between the NK cell and target. One aspect of NK cell synapses which has not been studied extensively is their sensitivity to the mechanical properties of the target. Diseased cells undergo alterations in cytoskeletal structure which manifest as a change in cellular stiffness. How this impacts the assembly and function of the NK cell synapse is currently unclear. Here, we set out to establish how target stiffness impacts NK cell cytotoxicity. Cell-sized microbeads were synthesised from sodium alginate to produce low (9 kPa), medium (34 kPa) or high (254 kPa) stiffness surrogate targets. These were coated with antibodies targeting the integrin LFA-1 and the activating receptors NKp30 and CD16. Using confocal imaging, the interactions of NK cells and targets of different stiffness were studied in 3D. A larger proportion of NK cells degranulated against stiff targets. Consistent with this, polarisation of the microtubule-organising centre (MTOC) and cytotoxic granules were dependent on target stiffness, as both occurred less frequently against soft targets. Thus, using a model system in which target cells of varied stiffness were modelled by gel microbeads, we demonstrate that NK cell responses are impaired against soft targets. A second aspect of NK cell cytotoxicity which remains unclear is the effect of the local microenvironment on the assembly of the immune synapse. Our knowledge of NK cell biology has largely relied on NK cells from the peripheral blood. Recently, it has become clear that tissue localisation profoundly affects the phenotype and function of NK cells. Lung NK cells are poorly lytic towards targets, but the cause of this hypofunctionality is not well understood. In parallel, we set out to understand why lung-derived NK cells cannot effectively kill targets. To address this, NK cells were isolated from matched human lung and blood samples. Using confocal imaging, the formation of synapses between lung NK cells and their targets was examined. Assembly of the immune synapse was unaffected by tissue localisation; however, polarisation of the MTOC and granules were impaired in NK cells from the lung. In addition, preliminary data indicated smaller mitochondria in lung NK cells. Taken together, our results add to our understanding of lymphocyte mechanosensitivity, and highlight the importance of the microenvironment in determining the NK cell functional response.

Date of Award	31 Dec 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Daniel Davis (Supervisor) & Tracy Hussell (Supervisor)