Ageing SLOw: Modelling Ageing in Secondary Lymphoid Organs in vitro

摘要动物模型未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1。传染病是消耗率最高的疾病之一，候选疫苗进入市场的平均概率为6%2，从临床前阶段开发的成本估计为3.74亿GB-1.5BN3。动物模型被用于测试和研究获得性免疫反应，但它们缺乏人类免疫的基本特征。更重要的是，我们从COVID19大流行中了解到，使用疫苗来保护65岁以上的人不如在年轻年龄组4有效。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，并在接种疫苗后无法产生持久的免疫力。对于季节性流感，这是研究最多的病原体之一，疫苗在老年人中的效力从0-50%5不等，导致NHS6的二级医疗费用为100 Mpa，90%的死亡是由65岁以上的感染造成的。然而，动物模型不能解释与年龄相关的免疫变化。导致几乎没有专门针对这一高危年龄段的疫苗。NAT的发展有可能推动与人类相关的疫苗开发，增加在老年人口中开发有效疫苗的机会。次级淋巴器官(SLO)、淋巴结和扁桃体是安装免疫反应和存储免疫记忆的地方。使它们成为模拟疫苗反应的理想器官。最近的研究表明，老年人对接种疫苗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--即基质支持细胞和细胞外基质(ECM)9决定的。目前建立SLO模型的方法依赖于免疫细胞在液体培养中自我聚集的能力，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组件并概括3D基质。然而，使用水凝胶对相关NAT进行生物工程是有局限性的。虽然合成水凝胶(如聚乙二醇；聚乙二醇)具有高度的重复性，生物力学性能，如硬度和粘度可以精确控制，但它们缺乏生物活性。此外，虽然天然材料(如胶原蛋白)具有极好的生物活性，但它们缺乏必要的重复性和生物力学的调节11。合成-生物杂化水凝胶(如聚乙二醇胶原)正在兴起--这些是可再生的材料，具有高生物活性和可调性12，13。因此，它们为NAT的开发提供了解决方案。在这个提案中，我将使用人类细胞和合成生物水凝胶来开发SLO的NAT模型。我的目标是：1)开发模拟健康和衰老微环境的SLO有机化合物；2)了解SLO微环境中的生物力学变化如何影响免疫功能；3)研究是否可以针对老年SLO的机械免疫机制来增强老年人的免疫反应。通过这项建议，我将使用有机类化合物和合成生物杂化水凝胶，以及广泛的系统，如活细胞成像和单细胞纳米压痕。此外，布里渊显微镜等新技术被评为2022年十大改变游戏规则的技术之一，因为它能够非侵入性地测量3D组织的生物力学14。有了这些，我将对SLO进行建模，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.