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

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.

We aim to determine how mechanical forces at the single-molecule level regulate the ability of naïve B cells to recognise and respond to antigen. The activation of antigen-specific B cells from the naïve repertoire leads to the production of neutralising antibodies, which are essential for protection against disease. There is growing evidence that mechanical forces at the single-molecule level influence the sensitivity and specificity of B cell responses, but until recently it has not been possible to unravel how forces transmitted to individual antigen binding receptors regulate the onset and strength of intracellular signalling. Here we 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 naïve 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 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.

Academic: We aim to combine single-molecule fluorescence and calcium imaging modalities with nanoscopic mechanosensors to generate a quantitative understanding of how B cells recognise and discriminate antigens at the earliest stage of the B cell response. The methods developed can easily be adapted to investigate virtually any receptor-ligand interaction in any cell, and the multiparameter data set can be used to develop mathematical models to predict B cell behaviour given a set of input signals.

Training: The PDRA to be recruited will receive continual cross-disciplinary training and mentoring in single-molecule biophysics and B cell immunology, which will aid their progression toward either an independent group leader position or a career in industry. The PDRA will receive academic training through co-authoring papers, grants, and reviews, and by presenting their work at national and international conferences in both the life and physical sciences. Research staff at King's are encouraged to take 10 days of personal and professional training each year. To facilitate this, King's offers a number of face-to-face, online learning, and webinar courses that will be available to the PDRA that are offered through the Centre for Doctoral Studies, the Researcher Development Programme, and the Centre for Research Staff Development. These courses cover a wide range of topics including Communication and Impact, Writing and Publishing, Statistics and Data Management, and Grant Writing. King's also has a career consultant who runs a series of workshops focused on a wide range of issues including career choices and academic career progression.

The project described here will generate numerous opportunities for student engagement from different backgrounds to interdisciplinary research in the life and physical sciences. The PI is the programme coordinator for a new undergraduate programme called Physics with Biophysics and has close associations with the MRes program Molecular Biophysics for Medical Sciences in the Randall Centre for Cell and Molecular Biophysics. Both BSc and MRes students will have the opportunity to conduct research projects in the PI's laboratory.

General Societal and Economical Impact: The societal benefits of this project are ultimately human health. The fundamental basis for the immune response lies at the single molecule level, involving an individual receptor-antigen engagement event. While classical described as a 'biological question', there is increasing evidence that mechanical forces also play a role. Biophysical methods, including high resolution optical microscopy combined with nanomechanical measurements, will be key to understanding the underlying physical basis of the onset of the immune response. Through the experiments described in this process we will begin to unravel the molecular mechanisms underlying how mechanical forces at the level of individual bonds between the B cell receptor and antigen regulate the sensitivity and specificity of the B cell response.