Ageing SLOw: Modelling Ageing in Secondary Lymphoid Organs in vitro

动物模型不能预测人类的治疗反应是一个主要问题。然而，非动物技术（NAT）正在成为一种可行的解决方案。目前缺乏与人类相关的临床前模型，导致注定失败的药物开发成本高昂，患者获得的药物更少。传染病的流失率最高，候选疫苗的平均市场进入概率为6%2，从临床前阶段的开发成本估计为3.74亿英镑至15亿英镑3。动物模型用于测试和研究适应性免疫反应，但它们缺乏人类免疫的基本特征。更重要的是，我们从COVID-19大流行中了解到，使用疫苗保护65岁以上的人群不如年轻人群有效。事实上，免疫功能随着年龄的增长而下降，导致感染易感性增加，接种疫苗后无法产生持久的免疫力。对于季节性流感（研究得最好的病原体之一），老年人的疫苗功效范围为0-50%5，导致NHS的二级医疗费用为100 Mpa 6，90%的死亡率是由65岁以上的感染引起的7。然而，动物模型不能解释年龄相关的免疫变异8。导致很少有专门针对这一高危年龄组的疫苗。NAT的开发有可能推动人类相关疫苗的开发，增加开发对老年人群有效的疫苗的机会。这将反过来减轻NHS的财政负担和能力压力。次级淋巴器官（SLO），淋巴结和扁桃体是免疫反应安装和免疫记忆存储的场所。使它们成为模拟疫苗反应的理想器官。最近，研究表明，老年人对疫苗接种的不良反应不是由循环免疫细胞的年龄决定的，而是由老化的SLO微环境决定的-即基质支持细胞和细胞外基质（ECM）9。目前模拟SLO的方法依赖于免疫细胞在液体培养物中自我聚集的能力，并且缺乏这些关键微环境成分的结合10。水凝胶提供了一种理想的解决方案，它们可以掺入ECM组分并重现3D基质。然而，使用水凝胶对相关NAT进行生物工程改造存在局限性。虽然合成水凝胶（例如聚（乙二醇）; PEG）是高度可再现的，并且可以精确地控制生物力学性质，例如刚度和粘度，但它们缺乏生物活性。此外，虽然天然材料（例如胶原蛋白）具有优异的生物活性，但它们缺乏必要的生物力学再现性和调整。合成-生物混合水凝胶（例如PEG-胶原蛋白）正在出现-这些是可再生的材料，具有高生物活性和可调性12，13。因此，他们提供了一个解决方案，NAT的发展。在这个建议中，我将开发NAT模型的SLO使用人类细胞和合成生物水凝胶。我的目标是：1）开发模拟健康和老化微环境的SLO类器官; 2）了解SLO微环境中的生物力学变化如何影响免疫功能，3）研究机械免疫机制是否可以在老年SLO中靶向，以增强老年人的免疫反应。在整个提案中，我将使用类器官和合成生物混合水凝胶，具有广泛的系统，例如活细胞成像和单细胞纳米压痕。以及新方法，如布里渊显微镜，由于其能够无创测量3D组织的生物力学，被评为2022年十大改变游戏规则的技术之一14。有了这些，我将模拟SLO并监测SLO微环境中的衰老和疫苗相关的机械免疫学变化。

SummaryThe failure of animal models to predict therapeutic responses in humans is a major problem. However, non-animal technologies (NATs) are emerging as a viable solution. The current lack of human-relevant preclinical models leads to costly development of drugs destined to fail and fewer drugs reaching patients1. Infectious diseases have one of the highest attrition rates, vaccine candidates have an average market entry probability of 6%2, and development from the pre-clinical stage costs an estimated £374M-£1.5BN3. Animal models are used to test and study adaptive immune responses, yet they lack essential features of human immunity. What's more, we know from the COVID19 pandemic, that using vaccines to protect over 65s is less effective than in younger age groups4. Indeed, immune function declines with age, leading to increased susceptibility to infection and failure to generate long-lasting immunity after vaccination. For seasonal influenza, one of the best studied pathogens, vaccine efficacy in the elderly ranges from 0-50%5, resulting in £100Mpa in secondary healthcare costs to the NHS6, and 90% of mortalities result from infection in over 65s7. Yet animal models cannot account for age-associated immune variation8. Resulting in few vaccines aimed specifically at this high-risk age group. The development of NATs has the potential to drive human-relevant vaccine development, increasing the chance of developing vaccines efficacious in the elderly population. This would, in-turn, relieve financial burden and capacity pressures on the NHS.Secondary lymphoid organs (SLOs), lymph nodes and tonsils, are the sites where immune responses are mounted and where immune memory is stored. Making them the ideal organ to model vaccine responses. Recently, it was shown that poor response to vaccination in the elderly is dictated not by the age of circulating immune cells, but by the aged SLO microenvironment - i.e. stromal support cells and extracellular matrix (ECM)9. Current approaches to model SLOs rely on the ability of immune cells to self-aggregate in liquid culture and lack incorporation of these critical microenvironment components10. Hydrogels provide an ideal solution, they can incorporate ECM components and recapitulate 3D stroma. Yet there are limitations to using hydrogels to bioengineer relevant NATs. While synthetic hydrogels (e.g. poly (ethylene glycol); PEG) are highly reproducible and biomechanical properties, such as stiffness and viscosity, can be precisely controlled, they lack biological activity. Also, while natural materials (e.g. collagen) have excellent bioactivity, they lack the necessary reproducibility and tuning of biomechanics11. Synthetic-biological hybrid hydrogels (e.g. PEG-collagen) are emerging - these are reproducible materials, with high bioactivity and tuneability12,13. Hence, they provide a solution for NAT development.In this proposal, I will develop NAT models of SLOs using human cells and synthetic-biological hydrogels. I aim to: 1) Develop SLO organoids that mimic healthy and aged microenvironments; 2) Understand how biomechanical changes in the SLO microenvironment affect immune function, and 3) Investigate if mechano-immunological mechanisms can be targeted in aged SLOs to enhance immune responses in the elderly.Throughout this proposal I will use organoids and synthetic-biological hybrid hydrogels, with an extensive range of systems, such as live cell imaging and single cell nanoindentation. As well as new approaches, such as Brillouin microscopy, voted one of the top 10 game-changing technologies of 2022 due to its ability to non-invasively measure the biomechanics of 3D tissues14. With these, I will model SLOs and monitor ageing and vaccine-related mechano-immunological changes in SLO microenvironments.