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

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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.