Early events in B cell activation.

B cell activation is initiated by the ligation of the B cell receptor (BCR) with antigen and ultimately results in the production of protective antibodies against potentially pathogenic invaders. Here we review recent literature concerned with the spatiotemporal dynamic characterization of the early molecular events of B cell activation, including the initiation of BCR triggering, the formation of BCR microclusters, and the dynamic regulation of BCR signaling. Because these events involve the considerable reorganization of molecules within the membrane, an important role for the cytoskeleton is emerging in the regulation of B cell activation. At each stage we highlight the role of the cytoskeleton, establishing its pivotal position during the initiation and regulation of B cell activation.

CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand.

Here we describe the spatiotemporal architecture, at high molecular resolution, of receptors and signaling molecules during the early events of mouse B cell activation. In response to membrane-bound ligand stimulation, antigen aggregation occurs in B cell antigen receptor (BCR) microclusters containing immunoglobulin (Ig) M and IgD that recruit the kinase Syk and transiently associate with the coreceptor CD19. Unexpectedly, CD19-deficient B cells were significantly defective in initiation of BCR-dependent signaling, accumulation of downstream effectors and cell spreading, defects that culminated in reduced microcluster formation. Hence, we have defined the dynamics of assembly of the main constituents of the BCR 'signalosome' and revealed an essential role for CD19, independent of the costimulatory molecule CD21, in amplifying early B cell activation events in response to membrane-bound ligand stimulation.

B cell receptor-mediated antigen gathering requires ubiquitin ligase Cbl and adaptors Grb2 and Dok-3 to recruit dynein to the signaling microcluster.

The B cell receptor (BCR) mediates B cell antigen gathering and acquisition for presentation to T cells. Although the amount of antigen presentation to T cells determines the extent of B cell activation, the molecular mechanisms underlying antigen gathering remain unexplored. Here, through a combination of high-resolution imaging, genetics and quantitative mass spectrometry, we demonstrate that adaptors Grb2 and Dok-3, and ubiquitin ligase Cbl in signaling BCR microclusters mediate association with the microtubule motor dynein. Furthermore, we visualize the localization and movement of these microclusters on the underlying microtubule network. Importantly, disruption of this network or diminished dynein recruitment in Grb2-, Dok-3-, or Cbl-deficient B cells, does not influence microcluster formation or actin-dependent spreading, but abrogates directed movement of microclusters and antigen accumulation. Thus we identify a surprising but pivotal role for dynein and the microtubule network alongside Grb2, Dok-3, and Cbl in antigen gathering during B cell activation.

The antigen expressway: follicular conduits carry antigen to B cells.

In this issue of Immunity, Roozendaal et al. (2009) visualize a conduit system within the lymph node follicle that allows transport of small antigens and chemokines from the subcapsular sinus to follicular B cells.

New insights into the early molecular events underlying B cell activation.

The appropriate activation of B cells is critical for the development and operation of immune responses and is dependent on the extensive coordination of intra- and intercellular communications in response to antigen stimulation. An accurate description of the B cell-activation process requires investigation of these interactions within their correct cellular context both at high resolution and in real time. Here, we discuss a number of recent studies that have offered insight into the early molecular events of B cell activation. We suggest that segregation within the B cell membrane triggers localized cytoskeleton reorganisation and signaling, allowing the formation of B cell receptor (BCR) microclusters. These BCR microclusters are the sites for the coordinated recruitment of the signalosome and are propagated during B cell spreading. We discuss the recent identification of a critical role for CD19 in the B cell response to membrane-bound antigen and suggest a mechanism involving BCR microclusters by which it mediates its stimulatory function. Finally, we consider research that has taken advantage of recent technological advances in multiphoton microscopy that have allowed its application to the investigation of the dynamics of membrane-bound antigen presentation and subsequent B cell activation in lymph nodes in vivo.

Visualizing a role for the actin cytoskeleton in the regulation of B-cell activation.

Appropriate activation of B cells is required for mounting protective humoral immune responses. B-cell activation is initiated following specific recognition of antigen by the B-cell receptor (BCR) and results in the generation of antibody-secreting plasma cells and long-lived memory cells. Initial imaging approaches revealed that B cells undergo dramatic molecular and morphological reorganizations following recognition of antigen. A number of these studies pointed to a role for the underlying cytoskeleton in regulating early events of B-cell activation. More recently, groundbreaking advances in imaging technologies have enabled direct visualization of the role for the cytoskeleton in regulating events at the B-cell membrane. Indeed, we have demonstrated that an ezrin-defined actin network shapes BCR diffusion and signaling both in the resting state and following antigen-induced activation. Importantly, alongside these in vitro imaging approaches, it has been demonstrated that mutations in cytoskeleton regulators such as CD19, dedicator of cytokinesis 8 (DOCK8), and Wiskott-Aldrich syndrome protein (WASp) are often associated with antibody deficiency syndromes in humans, establishing the importance of cytoskeleton reorganizations in conferring effective adaptive immunity.

B cell receptor-mediated uptake of CD1d-restricted antigen augments antibody responses by recruiting invariant NKT cell help in vivo.

Highly regulated activation of B cells is required for the production of specific antibodies necessary to provide protection from pathogen infection. This process is initiated by specific recognition of antigen through the B cell receptor (BCR), leading to early intracellular signaling followed by the late recruitment of T cell help. In this study we demonstrate that specific BCR uptake of CD1d-restricted antigens represents an effective means of enhancing invariant natural killer T (iNKT)-dependent B cell responses in vivo. This mechanism is effective over a wide range of antigen affinities but depends on exceeding a tightly regulated avidity threshold necessary for BCR-mediated internalization and CD1d-dependent presentation of particulate antigenic lipid. Subsequently, iNKT cells provide the help required for stimulating B cell proliferation and differentiation. iNKT-stimulated B cells develop within extrafollicular foci and mediate the production of high titers of specific IgM and early class-switched antibodies. Thus, we have demonstrated that in response to particulate antigenic lipids iNKT cells are recruited for the assistance of B cell activation, resulting in the enhancement of specific antibody responses. We propose that such a mechanism may operate to potentiate adaptive immune responses against pathogens in vivo.

Visualizing the molecular and cellular events underlying the initiation of B-cell activation.

The appropriate activation of B cells is critical for the development of effective immune responses. B cell activation is initiated following the engagement of the B cell receptor (BCR) with specific antigen. The spatiotemporal characterization of the ensuing molecular and cellular events has been the subject of recent high-resolution imaging investigations. In this review we highlight information gathered thus far concerning the initial processes underlying the activation of B cells. First, we consider studies that have offered new insights into the early molecular events that occur within the B cell prior to formation of the immunological synapse. As such, BCR-microclusters formed on engagement with antigen have been identified as the sites of active signaling and assembly of "microsignalosomes." Furthermore, signaling through these "microsignalosomes" is propagated and enhanced through B cell spreading in response to membrane-antigen in a CD19-dependent manner. Finally, we discuss a number of multiphoton microscopy studies that have enabled dynamic characterization of the initial encounters between B cells and antigen in vivo. These investigations visualize the presentation of larger antigens to B cells via cell-mediated strategies, involving macrophages in the subcapsular sinus and dendritic cells in the paracortex.

Microsignalosomes: spatially resolved receptor signalling.

B-cells are a critical component of the adaptive immune system. As such, B-cells survey the body and mount appropriate protective responses to pathogen-derived antigens, resulting in the production of specific antibodies and induction of immunological memory. Given the effectiveness of these responses in selectively eliminating pathogenic infections, it is clear that the processes underlying antigen-induced B-cell activation must be highly regulated. Somewhat surprisingly given the specialized function of these immune cells, the BCR (B-cell receptor) functions similarly to receptors of the tyrosine kinase family that are commonplace in biology, as BCR ligation with antigen leads to B-cell proliferation and differentiation. In the Lymphocyte Interaction Laboratory, we are particularly interested in characterizing the very early molecular events underlying B-cell activation using a combination of cutting-edge high-resolution and in vivo imaging techniques.

The intrinsic flexibility of IgE and its role in binding FcepsilonRI.

The interaction between IgE and its high affinity cellular receptor (FcepsilonRI) is an essential step in the development of allergic responses. Studies have identified the third constant domain of IgE (Cepsilon3) as the receptor binding region. The Cepsilon3 domain has unusual structural features; it was found to be a 'molten globule' structure in an isolated form, only assuming a well structured form upon binding to FcepsilonRI. The conformational flexibility intrinsic to the receptor binding portion of the molecule may be useful to IgE in allowing the large allosteric changes postulated to be required for FcepsilonRI engagement. If allosteric inhibitors can be developed then the dynamic properties of the Cepsilon3 domain may provide opportunities for therapeutic intervention in allergic disorders.

Catalytic folding of the Cepsilon3 domain by its high affinity receptor.

The interaction of immunoglobulin E (IgE) with its cellular receptor FcepsilonRIalpha is a central regulator of allergy. Structural studies have identified the third domain (Cepsilon3) of the constant region of epsilon heavy chain as the receptor binding region. The isolated Cepsilon3 domain is a "molten globule" that becomes structured upon binding of the FcepsilonRIalpha ligand. In this study, fluorescence and nuclear magnetic resonance spectroscopies are used to characterise the role of soluble FcepsilonRIalpha in the folding of the monomeric Cepsilon3 domain of IgE. Soluble FcepsilonRIalpha is shown to display characteristic properties of a catalyst for the folding of Cepsilon3, with the rate of Cepsilon3 folding being dependent on the concentration of the receptor.

Asymmetric segregation of polarized antigen on B cell division shapes presentation capacity.

During the activation of humoral immune responses, B cells acquire antigen for subsequent presentation to cognate T cells. Here we show that after mouse B cells accumulate antigen, it is maintained in a polarized distribution for extended periods in vivo. Using high-throughput imaging flow cytometry, we observed that this polarization is preserved during B cell division, promoting asymmetric antigen segregation among progeny. Antigen inheritance correlates with the ability of progeny to activate T cells: Daughter cells receiving larger antigen stores exhibit a prolonged capacity to present antigen, which renders them more effective in competing for T cell help. The generation of progeny with differential capacities for antigen presentation may have implications for somatic hypermutation and class switching during affinity maturation and as B cells commit to effector cell fates.

Monoclonal TCR-redirected tumor cell killing.

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)­mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

The cytoskeleton coordinates the early events of B-cell activation.

B cells contribute to protective adaptive immune responses through generation of antibodies and long-lived memory cells, following engagement of the B-cell receptor (BCR) with specific antigen. Recent imaging investigations have offered novel insights into the ensuing molecular and cellular events underlying B-cell activation. Following engagement with antigen, BCR microclusters form and act as sites of active signaling through the recruitment of intracellular signaling molecules and adaptors. Signaling through these "microsignalosomes" is propagated and enhanced through B-cell spreading in a CD19-dependent manner. Subsequently, the mature immunological synapse is formed, and functions as a platform for antigen internalization, enabling the antigen presentation to helper T cells required for maximal B-cell activation. In this review, we discuss the emerging and critical role for the cytoskeleton in the coordination and regulation of these molecular events during B-cell activation.

Antigen presentation to B cells.

B cells are capable of mounting responses to a bewildering range of potentially pathogenic antigens through the production of high-affinity antibodies and the establishment of immunological memory. Thus, regulated B-cell activation is critical for protection against a variety of bacterial and viral infections, as well as cancers. Here, we discuss a number of recent imaging studies that have provided new insights into the variety of mechanisms by which B-cell activation is initiated in the lymph node in vivo.

The who, how and where of antigen presentation to B cells.

A functional immune system depends on the appropriate activation of lymphocytes following antigen encounter. In this Review, we summarize studies that have used high-resolution imaging approaches to visualize antigen presentation to B cells in secondary lymphoid organs. These studies illustrate that encounters of B cells with antigen in these organs can be facilitated by diffusion of the antigen or by the presentation of antigen by macrophages, dendritic cells and follicular dendritic cells. We describe cell-surface molecules that might be important in mediating antigen presentation to B cells and also highlight the key role of B cells themselves in antigen transport. Data obtained from the studies discussed here highlight the predominance, importance and variety of the cell-mediated processes that are involved in presenting antigen to B cells in vivo.

Early events of B cell activation by antigen.

The activation of B cells confers long-lasting protection from a plethora of infectious diseases through the generation of plasma cells that produce high-affinity antibodies and memory cells. Engagement of the B cell receptor (BCR) with cognate antigen initiates intracellular signaling and subsequent internalization of antigen. Membrane-bound antigens are now considered the predominant forms that initiate B cell activation in vivo. We have shown that upon recognition of antigen on the surface of a presenting cell, the B cell undergoes a dramatic change in morphology characterized by rapid spreading followed by more prolonged contraction along the presenting surface. This two-phase response increases the amount of antigen that the B cell accumulates, internalizes, and subsequently presents to T cells. Thus, the spreading and contraction response shapes the outcome of B cell activation. We used a combination of planar lipid bilayers and total internal reflection fluorescence microscopy to investigate the early events that occur after engagement of the BCR and before B cell spreading. We observed the rapid formation of BCR-antigen microclusters, which we redefine as "microsignalosomes" because they mediate the coordinated recruitment of intracellular effectors, such as the kinases Lyn and Syk, the adaptor Vav, and phospholipase C-gamma2 (PLC-gamma2). We identified an essential role for the co-receptor CD19 in mediating spreading, and thus B cell activation, in response to membrane-bound antigen. Preliminary evidence suggests that the cellular morphology changes described in vitro are likely to occur upon recognition of antigen presented on the surface of macrophages in lymph nodes in vivo.

Regulation of integrin activation through the B-cell receptor.

Effective immune surveillance is absolutely dependent on the migration of lymphocytes throughout the body and on their successful recognition of specific antigens. Both of these functions rely on the capacity of integrins that are expressed on the surface of lymphocytes to respond in a highly regulated manner to a variety of chemokines and antigens. This Commentary is primarily concerned with the role of the B-cell integrins LFA-1 and VLA-4 in the antigen-recognition process, and summarises what is currently known about the molecular mechanisms of ;inside-out' integrin activation in response to B-cell-receptor stimulation. Recent investigations have identified Vav, PI3K and small GTPases as crucial regulators of the inside-out activation of B-cell integrins. These observations are of particular interest as they allude to an underlying mechanism by which B-cell-receptor-mediated signalling is linked to cytoskeleton reorganisation and subsequent integrin activation.

Phospholipase C-gamma2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen.

B cell receptor (BCR) recognition of membrane-bound antigen initiates a spreading and contraction response, the extent of which is controlled through the formation of signaling-active BCR-antigen microclusters and ultimately affects the outcome of B cell activation. We followed a genetic approach to define the molecular requirements of BCR-induced spreading and microcluster formation. We identify a key role for phospholipase C-gamma2 (PLCgamma2), Vav, B cell linker, and Bruton's tyrosine kinase in the formation of highly coordinated "microsignalosomes," the efficient assembly of which is absolutely dependent on Lyn and Syk. Using total internal reflection fluorescence microscopy, we examine at high resolution the recruitment of PLCgamma2 and Vav to microsignalosomes, establishing a novel synergistic relationship between the two. Thus, we demonstrate the importance of cooperation between components of the microsignalosome in the amplification of signaling and propagation of B cell spreading, which is critical for appropriate B cell activation.