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.

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 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.

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.

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.

The integrin leukocyte function-associated antigen-1 (LFA-1) is important in the promotion of B cell adhesion, thereby facilitating immunological synapse (IS) formation and B cell activation. Despite this significance, the associated signaling mechanisms regulating LFA-1 activation remain elusive. Here, we show that both isoforms of the small GTPase Rac expressed by primary B cells, Rac1 and Rac2, were activated rapidly downstream of Src-family kinases, guanine-nucleotide exchange factors Vav1 and Vav2, and phosphoinositide-3 kinase (PI3K) after BCR engagement. We identify Rac2, but not Rac1, as critical for B cell adhesion to intercellular adhesion molecule-1 (ICAM-1) and IS formation. Furthermore, B cells expressing constitutively active Rac2 are highly adhesive. We observe that Rac2-deficient B cells exhibit lower amounts of Rap1-GTP and severe actin polymerization defects, identifying a potential mechanism underlying their behavior. We postulate that this critical role for Rac2 in mediating B cell adhesion and IS formation might apply in all lymphocytes.

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-γ2 (PLCγ2), 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 PLCγ2 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.

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.