Specialised Architecture and Dynamics of Immune Synapses in B Cell Populations

B cells are specialised lymphocytes that are responsible for the production of antibodies during an immune response. When B cells bind membrane-presented antigen they form an immune synapse, which provides the context for coordinated B cell receptor (BCR) signalling and antigen internalisation, both of which are paramount for full B cell activation. Synapse formation has been well characterised in the largest B cell subset- follicular (FO) B cells, although little is known about how synapse formation is altered in less common B subsets, whose activation supports distinct aspects of the antibody response. Germinal center (GC) B cells develop from follicular B cells after antigen stimulation. They are confined to specialised sites in lymphoid organs, where they undergo somatic hypermutation and affinity improvement of their immunoglobulins. BCR signalling is speculated to be largely inactive in GC B cells, with antigen internalisation, processing and presentation to T cells driving their selection. However, there is limited knowledge of synapse formation, signalling and intracellular antigen trafficking in these cells. Using a newly developed high-content, large-scale fluorescent imaging platform we uncovered subset specific differences in synapse formation and mechanisms of antigen extraction in B cells of different subsets. Immunisation or infection-induced GC B cells showed the most striking difference, forming unique peripheral synapses where antigen was internalised by a distinct pathway independent of antigen movement to the centre of the contact. These cells still relied on proximal BCR signalling, but had defects in the NF-κB pathway, supporting a role for T-dependent selection of high affinity clones. GC cells produced higher tugging forces on the BCR than FO cells and were more effective at affinity discrimination. We conclude that unique biomechanical patterns control stringency of antigen binding in GC B cells, and propose that resultant effects on antigen presentation and availability of T cell help ultimately dictate GC B cell fate. Understanding requirements for the activation of GC B cells has applications in rational vaccine design, as well as for B cell malignancies. Therefore, these results have important implications for the future development of novel immunotherapies

WASp-dependent actin cytoskeleton stability at the dendritic cell immunological synapse is required for extensive, functional T cell contacts

The immunological synapse is a highly structured and molecularly dynamic interface between communicating immune cells. Although the immunological synapse promotes T cell activation by dendritic cells, the specific organization of the immunological synapse on the dendritic cell side in response to T cell engagement is largely unknown. In this study, confocal and electron microscopy techniques were used to investigate the role of dendritic cell actin regulation in immunological synapse formation, stabilization, and function. In the dendritic cell-restricted absence of the Wiskott-Aldrich syndrome protein, an important regulator of the actin cytoskeleton in hematopoietic cells, the immunological synapse contact with T cells occupied a significantly reduced surface area. At a molecular level, the actin network localized to the immunological synapse exhibited reduced stability, in particular, of the actin-related protein-2/3-dependent, short-filament network. This was associated with decreased polarization of dendritic cell-associated ICAM-1 and MHC class II, which was partially dependent on Wiskott-Aldrich syndrome protein phosphorylation. With the use of supported planar lipid bilayers incorporating anti-ICAM-1 and anti-MHC class II antibodies, the dendritic cell actin cytoskeleton organized into recognizable synaptic structures but interestingly, formed Wiskott-Aldrich syndrome protein-dependent podosomes within this area. These findings demonstrate that intrinsic dendritic cell cytoskeletal remodeling is a key regulatory component of normal immunological synapse formation, likely through consolidation of adhesive interaction and modulation of immunological synapse stability

PAISAJE DE Gran Canaria [Material gráfico]

Copia digital. Madrid : Ministerio de Educación, Cultura y Deporte. Subdirección General de Coordinación Bibliotecaria, 201

Cytoskeletal control of B cell responses to antigens.

The actin cytoskeleton is essential for cell mechanics and has increasingly been implicated in the regulation of cell signalling. In B cells, the actin cytoskeleton is extensively coupled to B cell receptor (BCR) signalling pathways, and defects of the actin cytoskeleton can either promote or suppress B cell activation. Recent insights from studies using single-cell imaging and biophysical techniques suggest that actin orchestrates BCR signalling at the plasma membrane through effects on protein diffusion and that it regulates antigen discrimination through the biomechanics of immune synapses. These mechanical functions also have a role in the adaptation of B cell subsets to specialized tasks during antibody responses

Plasma Membrane Sheets for Studies of B Cell Antigen Internalization from Immune Synapses.

Surrogate planar and membrane systems have been employed to study the architecture of immune synapses; however, they often do not recapitulate trans-synaptic extraction and endocytosis of ligands by the immune cells. Transendocytosis (or trogocytosis) of antigen from immune synapses is particularly critical for antigen processing and presentation by B cells. Here we describe a protocol for preparation of plasma membrane sheets (PMSs), which are flexible and fluid membrane substrates that support robust B cell antigen extraction. We show how to attach B cell antigens to the PMSs and how to investigate antigen extraction and endocytosis by fluorescent microscopy and computational image analysis. These techniques should be broadly applicable to studies of transendocytosis in a variety of cellular systems

WASp‐dependent actin cytoskeleton stability at the dendritic cell immunological synapse is required for extensive, functional T cell contacts

The immunological synapse is a highly structured and molecularly dynamic interface between communicating immune cells. Although the immunological synapse promotes T cell activation by dendritic cells, the specific organization of the immunological synapse on the dendritic cell side in response to T cell engagement is largely unknown. In this study, confocal and electron microscopy techniques were used to investigate the role of dendritic cell actin regulation in immunological synapse formation, stabilization, and function. In the dendritic cell‐restricted absence of the Wiskott‐Aldrich syndrome protein, an important regulator of the actin cytoskeleton in hematopoietic cells, the immunological synapse contact with T cells occupied a significantly reduced surface area. At a molecular level, the actin network localized to the immunological synapse exhibited reduced stability, in particular, of the actin‐related protein‐2/3‐dependent, short‐filament network. This was associated with decreased polarization of dendritic cell‐associated ICAM‐1 and MHC class II, which was partially dependent on Wiskott‐Aldrich syndrome protein phosphorylation. With the use of supported planar lipid bilayers incorporating anti‐ICAM‐1 and anti‐MHC class II antibodies, the dendritic cell actin cytoskeleton organized into recognizable synaptic structures but interestingly, formed Wiskott‐Aldrich syndrome protein‐dependent podosomes within this area. These findings demonstrate that intrinsic dendritic cell cytoskeletal remodeling is a key regulatory component of normal immunological synapse formation, likely through consolidation of adhesive interaction and modulation of immunological synapse stability. </p