Mechanical regulation of B cell antigen recognition at the single-molecule level

中和抗体是预防疾病的关键机制。它们是由适应性免疫系统对自然感染和疫苗接种的反应产生的。疫苗大大减少了人类的痛苦和与疾病有关的发病率，尽管一些对人类健康重要的疾病——包括艾滋病毒和流感——能够通过快速突变逃避适应性免疫反应。人们认为，抑制这些疾病将需要开发新的疫苗策略，诱导产生广泛中和的抗体，这些抗体针对致病表位，这些表位在许多突变体中都是保守的，并且随着时间的推移保持不变。目前的挑战是设计免疫原，引导适应性免疫反应向广泛中和抗体。抗体只由一群叫做B细胞的白细胞产生。人体内大约有100亿个B细胞，每个B细胞都会对一种独特的病原体做出反应，从而使免疫系统能够对大量潜在的免疫原性挑战做出反应。B细胞在称为B细胞受体（BCR）的膜结合抗体和称为抗原的致病表位之间的特异性细胞外结合相互作用后被激活。BCR-抗原结合诱导细胞内信号传导和B细胞活化程序，导致产生与BCR具有相同结合特异性的抗体。详细了解bcr -抗原结合导致B细胞活化和抗体产生的机制，对于合理开发针对高度可变病原体的新疫苗策略至关重要。目前的证据表明，B细胞与抗原结合，这些抗原首先被捕获并显示在其他称为抗原呈递细胞的免疫细胞表面。在BCR和表面上显示的抗原之间形成的键受到机械力的影响，这缩短了键的寿命。力也缩小了键寿命的分布，以便更好地区分与BCR有相似亲和力的抗原。施加力的大小和持续时间受细胞外机械刺激的影响，包括抗原呈递表面的分子张力和刚性，这已被证明会影响B细胞对抗原反应的敏感性和特异性。这些观察结果表明，可能通过设计具有特定机械特性的疫苗配方来调节B细胞的活化，从而引起所需的B细胞反应。要实现这一目标，需要了解BCR和抗原之间的结合是如何转化为细胞内信号传导的，以及力是如何调节信号传导从而调节B细胞结果的。在这里，我们将单分子荧光和钙成像与分子张力传感器相结合，以定量了解机械力如何在单分子水平上调节原代幼稚B细胞的抗原识别。我们的具体目标是：(1)确定细胞外与抗原的结合如何转化为细胞内信号传导；(2)确定抗原亲和力如何影响信号传导的启动；(3)确定底物硬度如何调节抗原密度和B细胞触发的亲和力阈值。这项工作在技术上是创新的，因为它结合了定量的、最先进的成像和生物物理工具来探索B细胞功能的机械调节。拟议的研究很重要，因为它们将导致对机械力如何调节B细胞识别和对抗原的反应的全面理解，这将有助于利用和控制B细胞活性以开发新疫苗。

Neutralising antibodies are a critical mechanism of protection against disease. They are produced by the adaptive immune system in response to both natural infection and vaccination. Vaccines have greatly reduced human suffering and disease related morbidity, although several important diseases for human health - including HIV and influenza - are able to evade the adaptive immune response through rapid mutation. It is thought that restraining these diseases will require the development of new vaccine strategies that induce the production of broadly neutralising antibodies that target pathogenic epitopes that are conserved across many mutants and remain constant over time. The current challenge is to design immunogens that steer the adaptive immune response toward broadly neutralising antibodies.Antibodies are produced exclusively by a population of white blood cells called B cells. Each of the approximately 10 billion B cells in the human body activates in response to a unique pathogen, giving the immune system the capability to respond to a huge range of potential immunogenic challenges. B cells become activated following specific, extracellular binding interactions between a membrane-bound antibody called the B cell receptor (BCR) and a pathogenic epitope called antigen. BCR-antigen binding induces intracellular signalling and a programme of B cell activation that leads to the production of antibodies that have the same binding specificity as the BCR. Developing a detailed understanding of the mechanisms by which BCR-antigen binding leads to B cell activation and antibody production is crucial for the rational development of new vaccine strategies that target highly mutable pathogens. Current evidence suggests that B cells engage antigens that are first captured and displayed on the surfaces of other immune cells called antigen-presenting cells. Bonds formed between the BCR and antigen displayed on a surface are subject to mechanical forces, which shorten the lifetime of the bonds. Forces also narrow the distribution of bond lifetimes to enable better discrimination between antigens with similar affinity for the BCR. The magnitude and duration of force application is influenced by extracellular mechanical stimuli including molecular tension and rigidity of the antigen-presenting surface, which have been shown to influence the sensitivity and specificity with which B cells respond to antigen. These observations suggest that it may be possible to regulate B cell activation by designing vaccine formulations with mechanical properties specifically tuned to elicit a desired B cell response. Achieving this goal will require understanding how the formation of bonds between the BCR and antigen is translated to intracellular signalling, and how forces act to modulate signalling and thus B cell outcomes.Here we will combine single-molecule fluorescence and calcium imaging with molecular tension sensors to build a quantitative understanding of how mechanical forces regulate antigen recognition by primary naive B cells at the single-molecule level. Our specific objectives are to: (1) determine how extracellular binding to antigen is translated into intracellular signalling; (2) determine how the initiation of signalling is influenced by antigen affinity; and (3) determine how substrate rigidity regulates antigen density and affinity thresholds for B cell triggering. This work is technically innovative because it incorporates quantitative, state-of-the-art imaging and biophysical tools to explore mechanical regulation of B cell function. The proposed studies are important because they will lead to a comprehensive understanding of how mechanical forces regulate B cell recognition and response to antigen, which will aid efforts to harness and control B cell activity for the development of new vaccines.