Ageing SLOw: Modelling Ageing in Secondary Lymphoid Organs in vitro

动物模型无法预测人类的治疗反应是一个主要问题。然而，非动物技术（NATs）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，导致药物开发成本高昂，但注定会失败，而且患者使用的药物也更少。传染病是损耗率最高的疾病之一，候选疫苗的平均市场进入概率为6%2，从临床前阶段开始的开发成本估计为3.74亿至1.5亿英镑3。动物模型被用来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岁以上人群的效果不如更年轻的年龄组4。事实上，免疫功能随着年龄的增长而下降，导致对感染的易感性增加，接种疫苗后无法产生持久的免疫力。季节性流感是研究得最好的病原体之一，疫苗对老年人的效力在0-50%之间5，给国家卫生服务体系造成了100兆帕的二级保健费用6,90%的死亡是由65岁以上的人感染造成的7。然而，动物模型无法解释与年龄相关的免疫变异。导致专门针对这一高危年龄组的疫苗很少。NATs的开发有可能推动与人类相关的疫苗开发，增加开发对老年人有效的疫苗的机会。反过来，这将减轻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，是免疫应答和免疫记忆储存的地方。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，而是由老年人的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中自聚集的能力，缺乏这些关键微环境成分的结合。水凝胶提供了一种理想的解决方案，它们可以结合ECM组件并重现3D基质。然而，使用水凝胶对相关的NATs进行生物工程还是有局限性的。而合成水凝胶（如聚乙二醇）；聚乙二醇（PEG）具有高度可重复性和生物力学性能，如刚度和粘度，可以精确控制，它们缺乏生物活性。此外，虽然天然材料（如胶原蛋白）具有出色的生物活性，但它们缺乏必要的生物力学可重复性和可调节性。合成生物杂化水凝胶（例如聚乙二醇-胶原蛋白）正在出现-这些是可再生材料，具有高生物活性和可调性12,13。因此，它们为NAT开发提供了一种解决方案。在本提案中，我将利用人类细胞和合成生物水凝胶开发slo的NAT模型。我的目标是：1)开发模拟健康和老年微环境的SLO类器官；2)了解SLO微环境的生物力学变化如何影响免疫功能；3)探讨老年SLO是否可以针对机械免疫机制增强老年人的免疫应答。在整个提案中，我将使用类器官和合成生物混合水凝胶，以及广泛的系统，如活细胞成像和单细胞纳米压痕。还有一些新方法，如布里渊显微镜，由于能够无创地测量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。有了这些，我将建立SLO模型，并监测SLO微环境中的衰老和疫苗相关的机械免疫变化。

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.