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

我们的免疫系统有多种武器来杀死入侵的病原体和流氓细胞。一个关键的武器是杀伤T细胞（也称为细胞毒性T淋巴细胞），这是一种白细胞，可以消除我们体内的病毒感染细胞和癌细胞。杀伤T细胞也可以从病人身上提取出来，并以所谓的CAR-T细胞的形式重新设计，以更有效地靶向肿瘤，最近在癌症免疫治疗中取得了一些最引人注目的成功。虽然杀伤T细胞的医学相关性是明确的，但重要的科学问题仍然是关于它们的功能。已知它们首先通过一种称为穿孔素的成孔蛋白的自组装穿孔靶细胞膜来杀死它们的靶标，这在一定程度上是由我们在Peter MacCallum癌症中心（墨尔本）的合作者的广泛工作以及我们自己与他们合作的工作所确定的（Leung， Hodel等人，Nat Nanotechnol 2017）。接下来，他们注入其他蛋白质，诱导目标细胞触发程序性细胞死亡（也称为细胞凋亡）。然而，有趣的是，这种有毒蛋白质组合的作用是单向的。也就是说，杀伤T细胞暴露在它们分泌的有毒蛋白质中，但在与靶细胞的接触中毫发无损，并且能够连续杀死多个靶细胞。通过过去几年在伦敦和墨尔本进行的纳米级生物物理学和细胞生物学实验，我们发现杀伤T细胞是通过其膜的物理特性来防止膜穿孔的。具体来说，我们发现更有序的脂质排列大大减少了穿孔蛋白与T细胞膜的结合；此外，膜上负电荷的局部暴露会破坏穿孔孔的形成（Rudd-Schmidt, Hodel et al.，提交）。与大多数令人兴奋的科学一样，这一发现带来了新的问题，我们希望通过这个博士项目来追求这些问题。特别是，我们将试图了解杀伤T细胞将更有序和带电的结构域引导到与靶细胞的免疫突触的机制。这个方向意味着在T细胞膜上分离成不同的脂质相，这些脂质相或多或少是有序和带电的。从物理上讲，有趣的问题是，尽管膜具有明显的流动性，但这种相分离是如何维持和指导的。为了回答这个问题，我们将对重建的模型膜和活T细胞进行纳米级成像实验/分析（原子力显微镜和超分辨率荧光显微镜），并通过开发和使用先进的图像分析（用于超分辨率显微镜数据）和流体膜相局部钉住的理论建模来解释我们的结果。

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.