The role of lipid order and charge in protecting killer T cells from suicide

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Our immune system has a wide range of weapons to kill invading pathogens and rogue cells. A key weapon is that of killer T cells (also called cytotoxic T lymphocytes), which are white blood cells that eliminate virus- infected and cancerous cells in our body. Killer T cells can also be extracted from patients and re-engineered in the form of so-called CAR-T cells to more effectively target tumours, having recently led to some of the most spectacular successes in cancer immunotherapy.While the medical relevance of killer T cells is unambiguous, important scientific questions remain about their function. They are known to kill their targets by first perforating the target cell membrane by self-assembly of a pore-forming protein called perforin, as established in part by extensive work of our collaborators in the Peter MacCallum Cancer Centre (Melbourne) and by our own work in collaboration with them (Leung, Hodel et al., Nat Nanotechnol 2017). Next, they inject other proteins that induce the target cells to trigger programmed cell death (also known as apoptosis). Intriguingly, however, the action of this toxic combination of proteins is unidirectional. That is, killer T cells are exposed to the toxic proteins they secrete and yet survive their encounters with target cells unscathed, and are capable to kill multiple target cells in succession.By joined nanoscale biophysics and cell biology experiments in London and Melbourne over the past years, we have discovered that killer T cells are protected against membrane perforation by the physical properties of their membranes. Specifically, we have found that more ordered lipid arrangements greatly reduce perforin binding to the T cell membranes; in addition, the local exposure of negative charge on the membrane disrupts perforin pore formation (Rudd-Schmidt, Hodel et al., submitted).As with most exciting science, this discovery leads to new questions that we aim to pursue with this PhD project. In particular, we will seek to understand the mechanisms by which killer T cells direct more ordered and charged domains to the immune synapse with the target cell. This direction implies a separation in the T cell membrane into different fluid lipid phases that are more/less ordered and charged. Physically, the intriguing question is how this phase separation is maintained and directed in spite of the noted fluidity of the membrane.To answer this question, we will carry out a combination of nanoscale imaging experiments/ analysis (atomic force microscopy and superresolution fluorescence microscopy) on reconstituted model membranes and live T cells, and interpret our results by developing and using advanced image analysis (for the superresolution microscopy data) and theoretical modelling of local pinning of fluid membrane phases.