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.
