Facundo D. Batista

B cells acquire antigen from target cells after synapse formation

Soluble antigen binds to the B-cell antigen receptor and is internalized for subsequent processing and the presentation of antigen-derived peptides to T cells. Many antigens are not soluble, however, but are integral components of membrane; furthermore, soluble antigens will usually be encountered in vivo in a membrane-anchored form, tethered by Fc or complement receptors. Here we show that B-cell interaction with antigens that are immobilized on the surface of a target cell leads to the formation of a synapse and the acquisition, even, of membrane-integral antigens from the target. B-cell antigen receptor accumulates at the synapse, segregated from the CD45 co-receptor which is excluded from the synapse, and there is a corresponding polarization of cytoplasmic effectors in the B cell. B-cell antigen receptor mediates the gathering of antigen into the synapse and its subsequent acquisition, thereby potentiating antigen processing and presentation to T cells with high efficacy. Synapse formation and antigen acquisition will probably enhance the activation of B cells at low antigen concentration, allow context-dependent antigen recognition and enhance the linking of B- and T-cell epitopes.

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.

Affinity Dependence of the B Cell Response to Antigen: A Threshold, a Ceiling, and the Importance of Off-Rate

Initiation and affinity maturation of the humoral immune response is driven by antigen interaction with BCR. To study how signaling and antigen presentation through BCR depend on antigen/BCR affinity, lysozyme-specific B cell transfectants were challenged with mutated lysozymes differing in their binding kinetics. For detectable triggering, the antigen/BCR complex needed a Ka > 10(6) M(-1) (dissociation half-life > approximately 1 s). Mutated lysozymes whose binding was below this threshold could nevertheless be presented if complexed with soluble antibody. Above the threshold, the concentration of antigen required to trigger a response decreased as the affinity (particularly dissociation half-life) increased. However, a plateau was reached at Kas > approximately 10(10) M(-1) (dissociation half-life > 0.5 hr), supporting the idea of a ceiling to affinity maturation.

RIF1 Is Essential for 53BP1-Dependent Nonhomologous End Joining and Suppression of DNA Double-Strand Break Resection

Summary The appropriate execution of DNA double-strand break (DSB) repair is critical for genome stability and tumor avoidance. 53BP1 and BRCA1 directly influence DSB repair pathway choice by regulating 5′ end resection, but how this is achieved remains uncertain. Here we report that Rif1−/− mice are severely compromised for 53BP1-dependent class switch recombination (CSR) and fusion of dysfunctional telomeres. The inappropriate accumulation of RIF1 at DSBs in S phase is antagonized by BRCA1, and deletion of Rif1 suppresses toxic nonhomologous end joining (NHEJ) induced by PARP inhibition in Brca1-deficient cells. Mechanistically, RIF1 is recruited to DSBs via the N-terminal phospho-SQ/TQ domain of 53BP1, and DSBs generated by ionizing radiation or during CSR are hyperresected in the absence of RIF1. Thus, RIF1 and 53BP1 cooperate to block DSB resection to promote NHEJ in G1, which is antagonized by BRCA1 in S phase to ensure a switch of DSB repair mode to homologous recombination.

LFA-1/ICAM-1 Interaction Lowers the Threshold of B Cell Activation by Facilitating B Cell Adhesion and Synapse Formation

The integrin LFA-1 and its ligand ICAM-1 mediate B cell adhesion, but their role in membrane-bound antigen recognition is still unknown. Here, using planar lipid bilayers and cells expressing ICAM-1 fused to green fluorescence protein, we found that the engagement of B cell receptor (BCR) promotes B cell adhesion by an LFA-1-mediated mechanism. LFA-1 is recruited to form a mature B cell synapse segregating into a ring around the BCR. This distribution is maintained over a wide range of BCR/antigen affinities (10(6) M(-1) to 10(11) M(-1)). Furthermore, the LFA-1 binding to ICAM-1 reduces the level of antigen required to form the synapse and trigger a B cell. Thus, LFA-1/ICAM-1 interaction lowers the threshold for B cell activation by promoting B cell adhesion and synapse formation.

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.

F-Actin Is an Evolutionarily Conserved Damage-Associated Molecular Pattern Recognized by DNGR-1, a Receptor for Dead Cells

Sterile inflammation can be initiated by innate immune recognition of markers of tissue injury termed damage-associated molecular patterns (DAMPs). DAMP recognition by dendritic cells (DCs) has also been postulated to lead to T cell responses to foreign antigens in tumors or allografts. Many DAMPs represent intracellular contents that are released upon cell damage, notably after necrosis. In this regard, we have previously described DNGR-1 (CLEC9A) as a DC-restricted receptor specific for an unidentified DAMP that is exposed by necrotic cells and is necessary for efficient priming of cytotoxic T cells against dead cell-associated antigens. Here, we have shown that the DNGR-1 ligand is preserved from yeast to man and corresponds to the F-actin component of the cellular cytoskeleton. The identification of F-actin as a DNGR-1 ligand suggests that cytoskeletal exposure is a universal sign of cell damage that can be targeted by the innate immune system to initiate immunity.

The Membrane Skeleton Controls Diffusion Dynamics and Signaling through the B Cell Receptor

Summary Early events of B cell activation after B cell receptor (BCR) triggering have been well characterized. However, little is known about the steady state of the BCR on the cell surface. Here, we simultaneously visualize single BCR particles and components of the membrane skeleton. We show that an ezrin- and actin-defined network influenced steady-state BCR diffusion by creating boundaries that restrict BCR diffusion. We identified the intracellular domain of Igβ as important in mediating this restriction in diffusion. Importantly, alteration of this network was sufficient to induce robust intracellular signaling and concomitant increase in BCR mobility. Moreover, by using B cells deficient in key signaling molecules, we show that this signaling was most probably initiated by the BCR. Thus, our results suggest the membrane skeleton plays a crucial function in controlling BCR dynamics and thereby signaling, in a way that could be important for understanding tonic signaling necessary for B cell development and survival.

Dock8 mutations cripple B cell immunological synapses, germinal centers and long-lived antibody production

To identify genes and mechanisms involved in humoral immunity, we did a mouse genetic screen for mutations that do not affect the first wave of antibody to immunization but disrupt response maturation and persistence. The first two mutants identified had loss-of-function mutations in the gene encoding a previously obscure member of a family of Rho-Rac GTP-exchange factors, DOCK8. DOCK8-mutant B cells were unable to form marginal zone B cells or to persist in germinal centers and undergo affinity maturation. Dock8 mutations disrupted accumulation of the integrin ligand ICAM-1 in the B cell immunological synapse but did not alter other aspects of B cell antigen receptor signaling. Humoral immunodeficiency due to Dock8 mutation provides evidence that organization of the immunological synapse is critical for signaling the survival of B cell subsets required for long-lasting immunity.

Identification of Bcl-6-dependent follicular helper NKT cells that provide cognate help for B cell responses

Lipid antigens trigger help from natural killer T cells (NKT cells) for B cells, and direct conjugation of lipid agonists to antigen profoundly augments antibody responses. Here we show that in vivo, NKT cells engaged in stable and prolonged cognate interactions with B cells and induced the formation of early germinal centers. Mouse and human NKT cells formed CXCR5+PD-1hi follicular helper NKT cells (NKTFH cells), and this process required expression of the transcriptional repressor Bcl-6, signaling via the coreceptor CD28 and interaction with B cells. NKTFH cells provided direct cognate help to antigen-specific B cells that was dependent on interleukin 21 (IL-21). Unlike T cell–dependent germinal centers, those driven by NKTFH cells did not generate long-lived plasma cells. Our results demonstrate the existence of a Bcl-6-dependent subset of NKT cells specialized in providing help to B cells.