Periodic Reporting for period 4 - BCELLMECHANICS (Regulation of antibody responses by B cell mechanical activity)
Reporting period: 2020-07-01 to 2022-05-31
The production of antibodies against pathogens is an effective mechanism of protection against a wide range of infections. However, some pathogens evade antibody responses by rapidly changing their composition. Designing vaccines that elicit antibody responses against invariant parts of the pathogen is a rational strategy to combat existing and emerging pathogens. Production of antibodies is initiated by binding of B cell receptors (BCRs) to foreign antigens presented on the surfaces of antigen-presenting cells. This binding induces B cell signalling and internalisation of the antigens for presentation to helper T cells. Although it is known that T cell help controls B cell expansion and differentiation into antibody-secreting and memory B cells, how the strength of antigen binding to the BCR regulates antigen internalisation remains poorly understood. As a result, the response and the affinity maturation of individual B cell clones are difficult to predict, posing a problem for the design of next-generation vaccines. Our aim is to develop an understanding of the cellular mechanisms that underlie critical B cell activation steps. Our laboratory has recently described that B cells use mechanical forces to extract antigens from antigen-presenting cells. We hypothesise that the application of mechanical forces tests BCR binding strength and thereby regulates B cell clonal selection during antibody affinity maturation and responses to pathogen evasion. The aims of this work were to (1) determine the magnitude and timing of the forces generated by B cells, (2) determine the role of the mechanical forces in affinity-dependent internalisation of antigens from immune synapses with antigen-presenting cells and (3) determine the role of antigen extraction from synapses with antigen-presenting cells in generating protective antibodies. The results of this work led to the development of DNA-nanosensors that measure pico-newton forces in live cell synapses and magnetic tweezer technology to measure bond-rupture dynamics in single molecules. We showed that germinal centre B cells use higher forces to extract antigens than naive B cells and that this promotes their ability to discriminate between weak and strong binding of antigens to their B cell receptors. We further identified the cytoskeletal mechanisms, depending on myosin IIa contractility and Arp2/3 actin polymerisation, which are critical for the application of B cell forces to extract antigens and produce antibodies. Further downstream, we characterised the endocytic pathways responsible for the delivery of the antigens into B cell processing compartments. Further work established techniques to knock out relevant genes in primary human cells for validation of these pathways. We also found that the immune synapse mechanics, and ligand discrimination, are regulated by the mechanical stiffness of antigen-presenting cells. Follicular dendritic cells in particular promote ligand discrimination by the B cell receptors. Finally, we identified the dynamics of antigen capture, retention and presentation to B cells by follicular dendritic cells in vivo after immunisation and showed how they are regulated by the receptor for antigen-immune complexes, CR1/2. Overall, this project established B cell and antigen-presenting cell mechanics as important factors regulating B cell receptor affinity discrimination and coupling to endocytic and presentation pathways required for B cell responses to antigens.
We developed new DNA-based nanosensors to measure cellular pulling forces. Using these sensors we showed that naïve B cells use low forces to extract antigens, whereas germinal centre B cells use high forces. This difference in mechanics of germinal centre B cells accompanies dramatically different architecture of their immune synapses and makes them more sensitive to antigen affinity. We conclude that germinal centre B cell mechanics likely contributes to selection of high affinity B cell clones during affinity maturation.
Improvements in the DNA sensors showed that B cells use mechanical forces to acquire antigens from live dendritic cells or follicular dendritic cells, two cell types involved in antigen presentation to B cells during antibody responses. In addition, force-mediated antigen extraction from antigen-presenting cells is regulated by the physical stiffness of the presenting cells. Dendritic cells are soft and allow extraction of even low affinity antigen via low forces. In contrast, follicular dendritic cells are stiff and promote stringent affinity discrimination. Thus, distinct mechanical properties of antigen-presenting cells promote different types of responses, with dendritic cells supporting sensitive activation of naïve cells, whereas follicular dendritic cells support affinity selection in germinal centres. The biophysical studies were supported by newly developed approaches to measuring single molecule bond rupture rates using optical tweezers, which were enhanced with holographic 3D imaging. Using genetic approaches, we established the importance of myosin IIa in B cell antigen extraction, proliferation and antibody production. We also described Arp2/3-generated actin foci that characterise the sites of antigen endocytosis using live cells and superresolution imaging. Through whole-genome CRISPR screens, we identified a novel endophilin-dependent endocytic pathway that is critical for B cell germinal centre responses. Further CRISPR-mediated gene targeting established genes important for these processes in human B cells as well. To understand the role of antigen presentation to B cells in vivo, we imaged the dynamics of antigen retention and presentation to B cells by follicular dendritic cells in draining lymph nodes after immunisation. These studies showed that a subpopulation of follicular dendritic cells located in the centre of B cell follicles and overlapping with germinal centres is responsible for the retention of antigens in the long term. Mechanistically, the central follicular dendritic cells express higher levels of CR1/2 receptors, which retain antigen in immune complexes. The presence of antigen on central follicular dendritic cells was required for efficient germinal centre responses and generation of plasma blasts.
These results were published in high-impact factor journals along with review and viewpoint articles, attracting 390 citations to date, and presented at several international meetings. We have also talked about our research in public to lay audiences at "Open days" of the Francis Crick Institute and in lay articles. The scientific progress lays foundation for future exploitation of the results in vaccine design, DNA-based nanotechnology and holographic imaging.
Improvements in the DNA sensors showed that B cells use mechanical forces to acquire antigens from live dendritic cells or follicular dendritic cells, two cell types involved in antigen presentation to B cells during antibody responses. In addition, force-mediated antigen extraction from antigen-presenting cells is regulated by the physical stiffness of the presenting cells. Dendritic cells are soft and allow extraction of even low affinity antigen via low forces. In contrast, follicular dendritic cells are stiff and promote stringent affinity discrimination. Thus, distinct mechanical properties of antigen-presenting cells promote different types of responses, with dendritic cells supporting sensitive activation of naïve cells, whereas follicular dendritic cells support affinity selection in germinal centres. The biophysical studies were supported by newly developed approaches to measuring single molecule bond rupture rates using optical tweezers, which were enhanced with holographic 3D imaging. Using genetic approaches, we established the importance of myosin IIa in B cell antigen extraction, proliferation and antibody production. We also described Arp2/3-generated actin foci that characterise the sites of antigen endocytosis using live cells and superresolution imaging. Through whole-genome CRISPR screens, we identified a novel endophilin-dependent endocytic pathway that is critical for B cell germinal centre responses. Further CRISPR-mediated gene targeting established genes important for these processes in human B cells as well. To understand the role of antigen presentation to B cells in vivo, we imaged the dynamics of antigen retention and presentation to B cells by follicular dendritic cells in draining lymph nodes after immunisation. These studies showed that a subpopulation of follicular dendritic cells located in the centre of B cell follicles and overlapping with germinal centres is responsible for the retention of antigens in the long term. Mechanistically, the central follicular dendritic cells express higher levels of CR1/2 receptors, which retain antigen in immune complexes. The presence of antigen on central follicular dendritic cells was required for efficient germinal centre responses and generation of plasma blasts.
These results were published in high-impact factor journals along with review and viewpoint articles, attracting 390 citations to date, and presented at several international meetings. We have also talked about our research in public to lay audiences at "Open days" of the Francis Crick Institute and in lay articles. The scientific progress lays foundation for future exploitation of the results in vaccine design, DNA-based nanotechnology and holographic imaging.
Our studies so far argue for an important role of B cell and antigen presenting cell mechanics that may be important for antibody responses. This new information opens a potential to modulate responses to vaccines in the future, for example by changing the physical properties of antigens and their presentation.