Zhengpeng Wan

B Cell Activation Is Regulated by the Stiffness Properties of the Substrate Presenting the Antigens

B lymphocytes are activated upon Ag sensing by BCRs. The substrate presenting the Ag can show different degrees of stiffness. It is not clear whether B cells can respond to changes in substrate stiffness. In this study we use high-resolution, high-speed live cell imaging techniques to capture the molecular events in B cell activation after the recognition of Ags tethered to polyacrylamide gel substrates with variable degrees of stiffness as quantified by Young’s modulus (2.6–22.1 kPa). We show that the initiation of B cell activation is extremely sensitive to substrate stiffness. B cells exhibit much stronger activation responses when encountering Ags tethered to substrates with a high degree of stiffness as measured by the accumulation of BCR, phospho-spleen tyrosine kinase, and phosphotyrosine molecules into the B cell immunological synapse. Ags tethered to stiff substrates induce the formation of more prominent BCR and phospho-spleen tyrosine kinase microclusters with significantly enhanced colocalization as compared with Ags tethered to soft substrates. Moreover, the expression of the B cell activation marker CD69 is enhanced in B cells encountering Ags on stiffer substrates. Through time-lapse live cell imaging, we find that the different responses of B cells to substrate stiffness are only demonstrated 5 min after BCR and Ag recognition. Using a series of cytoskeleton inhibitors, we determine that the mechanosensing ability of B cells is dependent on microtubules, and only mildly linked to the actin cytoskeleton. These results suggest the importance of the mechanical properties mediated by substrate stiffness in B cell activation.

The Scaffolding Protein Synapse-Associated Protein 97 Is Required for Enhanced Signaling Through Isotype-Switched IgG Memory B Cell Receptors

A scaffolding protein clusters B cell receptors to enable the rapid, high-titer antibody responses of memory B cells. Boosting Antibody Production The initial exposure of naïve B cells that have IgM B cell receptors (BCRs) on their surface to a foreign antigen produces a primary antibody response and generates memory B cells that have IgG BCRs, which respond to subsequent encounters with the same antigen by rapidly producing large amounts of antibodies. Liu et al. investigated differences in the signaling capacities of IgG and IgM BCRs and found that the scaffold protein SAP97 bound to IgG, but not IgM, BCRs at the immunological synapse, enabling BCR clustering and enhanced signaling. These findings may provide therapeutic targets to block enhanced BCR activation in autoimmune disease and in some B cell tumors. After their first encounter with a foreign antigen, naïve B cells that have immunoglobulin M (IgM) B cell receptors (BCRs) trigger the primary antibody response and the generation of memory B cells with IgG BCRs. When these memory B cells reencounter the same antigen, the cell surface IgG BCRs stimulate their rapid differentiation into plasma cells that release large amounts of IgG antibodies. We showed that the conserved cytoplasmic tail of the IgG BCR, which contains a putative PDZ (postsynaptic density 95/disc large/zona occludens 1)–binding motif, associated with synapse-associated protein 97 (SAP97), a PDZ domain–containing scaffolding molecule that is involved in controlling receptor density and signal strength at neuronal synapses. SAP97 accumulated and bound to IgG BCRs in the immunological synapses that formed in response to B cell engagement with antigen. Knocking down SAP97 in IgG+ B cells or mutating the putative PDZ-binding motif in the BCR tail impaired formation of the immunological synapse, initiation of IgG BCR signaling, and downstream activation of the mitogen-activated protein kinase p38. Thus, heightened B cell memory responses are encoded, in part, by a mechanism that involves SAP97 serving as a scaffolding protein in the IgG BCR immunological synapse.

Substrate stiffness regulates B-cell activation, proliferation, class switch, and T-cell-independent antibody responses in vivo

B cells use B‐cell receptors (BCRs) to sense antigens that are usually presented on substrates with different stiffness. However, it is not known how substrate stiffness affects B‐cell proliferation, class switch, and in vivo antibody responses. We addressed these questions using polydimethylsiloxane (PDMS) substrates with different stiffness (20 or 1100 kPa). Live cell imaging experiments suggested that antigens on stiffer substrates more efficiently trigger the synaptic accumulation of BCR and phospho‐Syk molecules compared with antigens on softer substrates. In vitro expansion of mouse primary B cells shows different preferences for substrate stiffness when stimulated by different expansion stimuli. LPS equally drives B‐cell proliferation on stiffer or softer substrates. Anti‐CD40 antibodies enhance B‐cell proliferation on stiffer substrates, while antigens enhance B‐cell proliferation on softer substrates through a mechanism involving the enhanced phosphorylation of PI3K, Akt, and FoxO1. In vitro class switch differentiation of B cells prefers softer substrates. Lastly, NP67‐Ficoll on softer substrates accounted for an enhanced antibody response in vivo. Thus, substrate stiffness regulates B‐cell activation, proliferation, class switch, and T cell independent antibody responses in vivo, suggesting its broad application in manipulating the fate of B cells in vitro and in vivo.

The activation of IgM- or isotype-switched IgG- and IgE-BCR exhibits distinct mechanical force sensitivity and threshold

B lymphocytes use B cell receptors (BCRs) to sense the physical features of the antigens. However, the sensitivity and threshold for the activation of BCRs resulting from the stimulation by mechanical forces are unknown. Here, we addressed this question using a double-stranded DNA-based tension gauge tether system serving as a predefined mechanical force gauge ranging from 12 to 56 pN. We observed that IgM-BCR activation is dependent on mechanical forces and exhibits a multi-threshold effect. In contrast, the activation of isotype-switched IgG- or IgE-BCR only requires a low threshold of less than 12 pN, providing an explanation for their rapid activation in response to antigen stimulation. Mechanistically, we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold. These results defined the mechanical force sensitivity and threshold that are required to activate different isotyped BCRs. DOI: http://dx.doi.org/10.7554/eLife.06925.001

The growth of B cell receptor microcluster is a universal response of B cells encountering antigens with different motion features

B lymphocyte cell senses and acquires foreign antigens through clonal distributed B cell receptors (BCRs) expressed on the surface of plasma membrane. The presentation formats of antigens are quite diverse. Based on their Brownian diffusion mobility, there are three forms: free mobile soluble antigens, lateral mobile membrane bound antigens, and fixed immobile antigens. Here, using high resolution high speed live cell imaging approaches, we provide evidence that BCR microclusters are formed on the surface of B cells shortly after B cell’s encountering of antigens with each format of motion features. Through high speed live cell imaging, we determine that these BCR microclusters show dynamic growth feature and by doing so function as the basic platforms for B cells to acquire the antigens. We propose that the formation and dynamic growth of BCR microcluster is a universal mechanism for B cell to response to antigens with diverse motion features.

Through an ITIM-Independent Mechanism the FcγRIIB Blocks B Cell Activation by Disrupting the Colocalized Microclustering of the B Cell Receptor and CD19

B cell activation is regulated through the interplay of the BCR with the inhibitory coreceptor FcγRIIB and the activating coreceptor CD19. Recent studies suggest that Ag-driven BCR microclusters are efficiently converted to a signaling active state on colocalization with CD19 microclusters. Using total internal reflection fluorescence microscopy–based, high-resolution, high-speed live-cell and molecule imaging approaches, we show that when co-ligated to the BCR, the FcγRIIB can inhibit B cell activation by blocking the colocalization of BCR and CD19 microclusters within the B cell immunological synapse. Remarkably, this inhibitory function of FcγRIIB is dependent not on its well-characterized ITIM-containing cytoplasmic domain, but its transmembrane domain. Indeed, human primary B cells from systemic lupus erythematosus patients homozygous for gene encoding the loss-of-function transmembrane domain mutant FcγRIIB-I232T fail to block the synaptic colocalization of the BCR with CD19, leading to dysregulated recruitment of downstream signaling molecule p-PI3K to membrane proximal signalosome. This inhibitory function of FcγRIIB in impairing the spatial-temporal colocalization of BCR and CD19 microclusters in the B cell immunological synapse may help explain the hyper-reactive features of systemic lupus erythematosus patient B cells in reported studies. These observations may also provide new targets for therapies for systemic autoimmune disease.

Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

Significance B-cell receptor (BCR) and antigen engagement induces several responses resulting in B-cell activation. However, it has been difficult to study these responses due to their dynamic nature. To solve this problem, a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP), was developed. B cells contacting caged-NP exhibited probing behaviors that are cell intrinsic with strict dependence on F-actin remodeling. B-cell probing behaviors were terminated within 4 s after the photoactivation of caged-NP. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. The analysis of temporally segregated single molecule images demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens. Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens.

Substrate stiffness governs the initiation of B cell activation by the concerted signaling of PKCβ and focal adhesion kinase

The mechanosensing ability of lymphocytes regulates their activation in response to antigen stimulation, but the underlying mechanism remains unexplored. Here, we report that B cell mechanosensing-governed activation requires BCR signaling molecules. PMA-induced activation of PKCβ can bypass the Btk and PLC-γ2 signaling molecules that are usually required for B cells to discriminate substrate stiffness. Instead, PKCβ-dependent activation of FAK is required, leading to FAK-mediated potentiation of B cell spreading and adhesion responses. FAK inactivation or deficiency impaired B cell discrimination of substrate stiffness. Conversely, adhesion molecules greatly enhanced this capability of B cells. Lastly, B cells derived from rheumatoid arthritis (RA) patients exhibited an altered BCR response to substrate stiffness in comparison with healthy controls. These results provide a molecular explanation of how initiation of B cell activation discriminates substrate stiffness through a PKCβ-mediated FAK activation dependent manner. DOI: http://dx.doi.org/10.7554/eLife.23060.001

How B cells remember? A sophisticated cytoplasmic tail of mIgG is pivotal for the enhanced transmembrane signaling of IgG-switched memory B cells.

Antibody memory is critical for protection against many human infectious diseases and is the basis for nearly all current human vaccines. Isotype switched immunoglobulin (Ig) G-expressing memory B cells are considered as one of the fundaments for the rapid, high affinity and high-titered memory antibody response. The detailed molecular mechanism of the enhanced activation of IgG-switched memory B cells upon BCR engagement with antigens has been an elusive question in immunology. In this review, we tried to discuss all the exciting new advances revealing the molecular mechanisms of the transmembrane signaling through mIgG cytoplasmic tail in IgG-switched memory B cells.

A New and Robust Method of Tethering IgG Surrogate Antigens on Lipid Bilayer Membranes to Facilitate the TIRFM Based Live Cell and Single Molecule Imaging Experiments

Our understanding of cell-cell interactions has been significantly improved in the past years with the help of Total Internal Reflection Fluorescence Microscope (TIRFM) in combination with an antigen presenting system supported by planar lipid bilayer (PLB) membranes, which are used to mimic the extensive receptor and ligand interactions within cell-cell contact interface. In TIRFM experiments, it is a challenge to uniformly present ligand molecules in monomeric format on the surface of PLB membranes. Here, we introduce a new and robust method of tethering IgG surrogate antigen ligands on the surface of Ni2+-containing PLB membranes. In this method, we use a modified D domain from staphylococcal protein A molecule that is fused with an N-terminus polyhistidine tag (H12-D-domain) to tether IgG surrogate antigens on Ni2+-containing PLB membranes. We systematically assessed the specificity and capability of H12-D-domain construct to capture IgG molecules from different species through live cell and single molecule TIRFM imaging. We find that these IgG surrogate antigens tethered by H12-D-domain show better lateral mobility and are more uniformly distributed on PLB membranes than the ones tethered by streptavidin. Neither IgM molecules, nor Fab or F(ab’)2 fragments of IgG molecules can be tethered on PLB membranes by H12-D-domain construct. These tethered IgG surrogate antigens strongly induce the formation and accumulation of signaling active antigen receptor microclusters within the immunological synapse in B or T lymphocyte cells. Thus our method provides a new and robust method to tether IgG surrogate antigens or other molecules fused with IgG Fc portion on PLB membranes for TIRFM based molecule imaging experiments.