T-Independent Antigens

• Not all antibody-producing responses need T cells; some B cell subsets can secrete antibodies to specific antigens without T-cell help.
• T-independent antibody responses are often triggered by antigens with polyvalent, repeating determinants shared among microbial species.
• B-1a, B-1b, and marginal zone B cells recognize many T-independent antigens.
• B-1a and B-1b cells secrete mainly IgM antibodies that are not subject to SHM.
• B-1 B cells are considered "innate-like" lymphocytes due to their shared antigens and oligoclonal antibody responses.
• Marginal zone B cells might receive help from cell types other than T cells.

T-Independent Antigens

• Nude mice, lacking body hair and with oversized ears, have a mutation in the Foxn1 gene, resulting in athymia and few mature T cells.
• Immunologists used nude mice to demonstrate that most protein antigens fail to elicit an antibody response in the absence of T cells.
• Carbohydrate antigens can generate antibody responses in athymic mice and are termed T-independent antigens.
• T-independent antigens elicit predominantly low-affinity antibody responses from B cells expressing mainly the IgM isotype because T-cell interactions are required for the induction of AID and hence for SHM.
• T-independent antigens are classified into two subclasses: TI-1 and TI-2.

TI-1 Antigens

• Lipopolysaccharide (LPS), expressed by gram-negative bacteria, is an example of a TI-1 antigen.
• TI-1 antigens bind to innate immune receptors on all B cells (including most B-2 B cells).
• At high antigen doses, all B cells bearing the relevant innate receptors can be mitogenically activated.
• The dramatic in vivo polyclonal TI-1 responses generated in response to high levels of gram-negative organisms can be catastrophic for an individual and are associated with the phenomenon of septic shock.
• A small minority of the antibodies produced will be able to bind directly to the TI-1 antigen because B-cell stimulation in this instance is occurring through the innate receptor (TLR4/MD-2/CD14).
• At lower doses, the innate immune receptors are unable to bind sufficient antigen to be stimulatory for the B cell; however, in those B cells that bind to the TI-1 antigen through their Ig receptors, the TI-1 antigen cross-links the Ig and innate receptors, thereby eliciting an oligoclonal B-cell response that remains independent of T cells.
• Under low-dose circumstances, all secreted antibodies will be specific for the TI-1 antigen.

TI-2 Antigens

• TI-2 antigens, such as capsular bacterial polysaccharides or polymeric flagellin, do not activate B cells via innate immune receptors and do not elicit a polyclonal response at high concentrations.
• TI-2 antigens activate B cells without T-cell help because they present antigenic determinants in a multivalent array, causing extensive BCR cross-linking.
• Most naturally occurring TI-2 antigens bind complement fragments C3d and C3dg.
• TI-2 antigens activate B cells by cross-linking BCR and CD21 (CR2) receptors on the B-cell surface.
• Marginal zone B cells are highly represented among B cells that bind TI-2 antigens through BCR and CD21 binding.
• TI-2 antigens can only partially stimulate B cells without help from other cells.
• Monocytes, macrophages, and dendritic cells facilitate B-cell responses to TI-2 antigens by secreting BAFF, promoting B-cell survival, maturation, and antibody secretion.
• While not necessary, T cells may enhance B-cell activation by TI-2 antigens by producing cytokines which stimulate antibody classes other than IgM.
• TI-2 antigens cannot stimulate immature B cells and do not act as polyclonal activators.
• Interactions with T cells, macrophages, monocytes, and neutrophils enhance both types of T-independent responses.

Novel Subclasses of B Cells

• Recent research has revealed multiple B-cell subpopulations differing in location, phenotype, and function.
• Transitional B-cell populations T1 and T2 represent temporal stages of B-cell development, while T3 appears to be an anergic subpopulation.
• B-1a, B-1b, B-2, and marginal zone B cells are different subpopulations of mature B cells with distinct locations and functional capacities.
• The T-dependent B-cell response is conducted by B-2 cells, also known as follicular B cells.
• B cells with specificity for T-independent antigens are divided into two major subtypes with differences in development, location, and markers.

B-1 B Cells

• B-1 B cells occupy a midpoint between innate and adaptive immune systems, located outside classical secondary lymphoid tissues.
• Lower diversity in antigen receptors compared to B-2 B cells characterizes B-1 B cells.
• Respond rapidly to antigen challenge with low-affinity responses primarily of the IgM class.
• Self-renewing in the periphery without continuous reseeding from bone marrow precursors.
• B-1 B cells were first described in 1983 with the discovery of CD5-bearing B cells.
• They appear before B-2 B cells during embryonic development.
• B-1 B cells make up about 5% of B cells in humans and mice but are the most abundant in the peritoneal cavity.
• They generate rapid responses to T-independent antigens, and most antibodies recognize common, repeated antigenic determinants expressed by gut and respiratory system bacteria.
• B-1 B cells do not undergo somatic hypermutation and primarily secrete IgM antibodies of relatively low affinity and less diversity.
• B-1 B cells are found at relatively high frequency among B cells during fetal and neonatal life.
• B-1b B cells were identified later, having functional characteristics of B-1 cells but lacking CD5 expression.
• Natural IgM antibodies, found in unimmunized animals, bind a broad spectrum of antigens with low affinity and high cross-reactivity, deriving mainly from B-1 B cells.
• These antibodies may display autoimmune reactivities, with interactions with self-antigens potentially important in B-1 B-cell development and function.
• B-1 B-cell antibody secretion is not dependent on T-cell help but can be enhanced by T cell–derived cytokines.
• Some B-1b cells may express certain attributes of B-2 cells, such as Ig class switching, low SHM levels, and production of long-lived memory cells with T-cell help.

Marginal Zone B Cells

• Marginal zone (MZ) B cells reside in the marginal zone of the spleen and respond to TI antigens.
• The subset's location allows rapid exposure to antigens carried to the spleen via the blood.
• Antigens presented to MZ B cells are captured by metallophilic or marginal zone macrophages in the marginal sinus, or by neutrophils or dendritic cells in the circulation.
• Differences in microvasculature between human and mouse spleens impact the structure and function of the MZ.
• In mice, blood flows slowly through the MZ, while in humans, blood drains directly into capillaries of the red pulp and perifollicular zone.
• Human MZ B cells consist largely of IgM+ memory cells, while mouse MZ cells are thought to represent a distinct B-cell lineage.
• Mouse MZ B cells are larger than follicular B cells, expressing higher levels of IgM, CD21, CD80, and CD86, but lower levels of IgD.
• They differentiate into plasmablasts more rapidly than follicular B cells.
• Elevated CD21 levels enable efficient binding to antigens conjugated to C3d or C3dg.
• MZ B cells are important in host protection against pathogens bearing TI-2 antigens.
• Effective cells in antigen presentation and efficient at activating memory CD4+ T cells.
• MZ B cells pick up antigen from metallophilic macrophages or from dendritic cells and neutrophils.
• BCR-mediated stimulation of MZ B cells leads to antibody secretion.
• They can shuttle antigens into the B-cell follicles if antigens are bound by complement receptors.
• MZ B cells exhibit long membrane extensions and rapid motility, facilitating antigen transport.
• Maintenance of MZ B cells depends on low-level signals through the BCR and survival signals such as BAFF.
• They derive originally from the transitional T2 B-cell population and have the capacity for self renewal in the periphery.
• Antigen stimulation results in movement from the MZ to bridging channels and red pulp, forming foci of plasmablasts and producing high levels of antigen-specific IgM.
• In antibody secretion, SHM, and CSR, MZ B cells may be helped by neutrophils and other cells, including T cells.
• Mouse marginal zone B cells are exposed directly to antigen entering the spleen and are an important first line of defense, with neutrophils participating in regulating their differentiation.
• B cells differentiate into plasma cells that produce antibodies, which bind to harmful organisms or molecules.
• These secreted antibodies have the same antigen-binding sites as the B-cell surface receptor molecules.
• Antibodies are proteins known as immunoglobulins that protect against pathogens like viruses, bacteria, and parasites.

Burnet's Clonal Selection Hypothesis

• Sir Frank Macfarlane Burnet's 1957 paper laid the foundation for B-cell clonal selection, activation, proliferation, and deletion.
• The clonal selection hypothesis states that the lymphocyte surface receptor molecule and the secreted antibody products have identical antigen-binding specificities.
• Stimulation of a single lymphocyte results in a clone of cells with the same receptor specificity as the original cell.
• Daughter cells within each clone secrete large amounts of specific antibodies.
• Some progeny cells remain viable after infection to neutralize secondary infections by the same pathogen.
• Immature B lymphocytes have immunoglobulin (Ig) receptors on their cell surfaces, all with identical specificity for an antigen.
• Upon antigen stimulation, the B cell matures, migrates to lymphoid organs, and replicates.
• Its clonal descendants bear the same receptor as the parental B cell and secrete antibodies with identical specificity for the antigen.
• More B cells bearing receptors for the stimulating antigen remain in the host after the immune response than before the antigenic challenge.
• These memory B cells can mount an enhanced secondary response.
• B cells with receptors for self-antigens are deleted during embryonic development.

Burnet's Predictions

• Predicted the generation of a vast array of antibody specificities.
• Postulated novel genetic mechanisms to create the antigen receptor repertoire, requiring a "randomization" of the coding responsible for part of the specification of gamma globulin molecules during early embryonic development.
• Predicted the need for clonal deletion in the developing B-cell population to eliminate cells bearing receptors specific for self-antigens.
• Awarded the Nobel Prize in 1960 with Peter Medawar for his clonal selection hypothesis and work on immunological tolerance.

B-Cell Responses

• Two major types of B-cell responses are elicited by structurally distinct types of antigens.
• T-dependent (TD) responses are generated following recognition of protein antigens and require CD4+ helper T cells.
• TD responses are mediated by B-2 B cells, which are responsible for high-affinity, memory B cells of secondary antibody responses.
• B-2 B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR), resulting in high-affinity antibodies of isotypes other than IgM.
• T-independent (TI) responses are directed toward multivalent or highly polymerized antigens and do not require T-cell help.
• TI-1 antigens bind to innate receptors on B cells, are mitogenic, and elicit a polyclonal, antibody-secreting response at high concentrations.
• TI-2 antigens are highly multivalent and bind only to Ig receptors, cross-linking a large fraction of the Ig receptors on the surface of a B cell to deliver an activation signal without T-cell help.
• Most T-independent responses are mediated by B-1 B cells and marginal zone B cells.

T-Dependent B-Cell Responses: Activation

• After maturation, B cells migrate to lymphoid follicles, where they can interact with antigen and become activated or recirculate through the blood and lymphatic systems.
• B-cell survival depends on B-cell activating factor (BAFF), a TNF family member cytokine secreted by lymphoid stromal cells and innate immune cells.
• Mature B cells unable to secure sufficient BAFF undergo apoptosis.
• Recirculating mature B cells have a half-life of approximately 4.5 months.
• At the start of a T-dependent B-cell response, the B cell binds antigen via its Ig receptors (signal 1).
• Some of the bound antigen is internalized, processed, and re-expressed in the form of peptides in the antigen-binding groove of MHC class II molecules.
• Signal 2 is provided by an activated T cell, which binds to the B cell through its antigen receptor and via interactions between CD40 and B7 on the B cell and CD40L (CD154) and CD28 on the activated TH cell.
• The T cell releases its activating cytokines (signal 3) directly into the T-cell/B-cell interface.
• The nature of the response is also affected by cytokines released by other cells in the vicinity of the antigen encounter.

Adoptive Transfer Experiments

• Experiments by Miller, Mitchell, and Mitchison in the 1960s using adoptive transfer demonstrated that B cells required "help" from T cells.
• Mice were lethally irradiated to eliminate their lymphocytes and then reconstituted with purified cells from genetically identical donors to generate an immune response.
• A mouse must receive cells from both the bone marrow and the thymus of a healthy donor animal to generate an antibody response.
• Thymus-derived cells were helper T cells, while bone marrow-derived, antibody-producing cells were mature B cells recirculating through the bone marrow.
• The antibody response to protein antigens required both B and T cells.

B-Cell Differentiation Pathways

• Contemporary work has shown that signals delivered from the T cell to the antigen-activated B cell stimulate the B cell to differentiate along one of three pathways.
• It can proliferate to form a "primary focus" of antibody-secreting plasma cells that provide the initial IgM antibodies of the primary response.
• It can develop directly into an IgM-bearing memory cell.
• It can enter the germinal center (GC) and undertake one of the most complex differentiation programs in biology.
• Following T-dependent stimulation with antigen, B cells may differentiate into antibody-producing plasma cells in the primary focus or into early, low-affinity memory cells or enter the follicles to participate in the germinal center reaction.

Antigen Encounter in Lymph Nodes and Spleen

• When antigen is introduced into the body, it is concentrated in peripheral lymphoid organs (spleen for blood-borne antigen, lymph nodes for tissue spaces).
• B cells recognize antigenic determinants on native, unprocessed antigens.
• Antigen is often covalently modified with complement fragments, and CR2 (CD21) on B cells binds complement-coupled antigen.
• The process of antigen sampling by B cells in the gut lymphoid system happens in the lymph nodes.

Mechanism of B-Cell Antigen Acquisition

• Soluble antigens picked up by the afferent lymphatic vessels flow into the subcapsular sinus cavity of the lymph node.
• Antigens with a molecular weight less than 70 kDa enter a system of conduits originating in the base of the subcapsular sinus (SCS).
• These conduits are produced by fibroblasts.
• Dendritic cells, macrophages, and B cells can access the antigens carried in these conduits by extending processes through the basement membrane.
• Conduits transport chemokines, attracting cells to the conduit contents, and terminate near high endothelial venules, which act as ports of entry for lymphocytes.

Large Antigen Entry

• Larger, more complex antigens are picked up by subcapsular sinus macrophages (SCSMs), a subpopulation of macrophages with limited phagocytic ability.
• SCSMs express high levels of cell-surface molecules able to bind and retain unprocessed antigen.
• Bacteria, viruses, particulates, and other complex antigens covalently linked to complement components are held by complement receptors on the surfaces of these macrophages.
• B cells in the lymph node follicles migrate to regions directly underlying the capsule and acquire antigen directly from the SCSMs, subsequently returning to the follicle.
• Antigens also bind to the surfaces of dendritic cells and follicular dendritic cells via complement and other receptors and can be passed from these cell types to B cells.

Immunization and Infection

• Immunization or infection with antigens previously encountered results in the formation of immune complexes of antigens and antibodies.
• These are picked up by Fc receptors on noncognate B cells (B cells whose native receptors are not specific for the antigens) and macrophages and enter the lymph nodes passively on these cells.
• Once in the lymph node, these antigen-transporting cells traffic to the follicles under the influence of chemokines, where their antigen can be recognized by B cells bearing the appropriate B-cell receptor (BCR).

Follicular Dendritic Cells

• The dendrites of follicular dendritic cells (FDCs) are studded with antigen-antibody complexes, retained on the surface through interaction with Fc or complement receptors.
• FDCs main function is to provide a reservoir of antigen for B cells to bind as they undergo mutation, selection, and differentiation during germinal center differentiation.
• The half-life of antigen on the surface of FDCs is relatively long.
• FDCs secrete factors that ensure the survival of B cells within the lymph node.
• Marginal zone B cells have also been implicated in antigen transport and presentation in the spleen.

Key Concepts

• Some low-molecular-weight antigens enter the lymph nodes via a leaky network of conduits that are sampled by follicular B cells.
• Higher molecular weight antigens are taken up first by Fc or complement receptors on subcapsular sinus macrophages or by similar receptors on B cells, dendritic cells, and circulating macrophages, and subsequently passed on to the B cells.

B-Cell Recognition of Cell-Bound Antigen

• Prior to antigen contact, most B-cell receptors (BCRs) are expressed on the B-cell surface in tiny nanoclusters.
• Interaction of BCRs with multivalent, cell-bound antigens induces a response of the B-cell membrane.
• A few BCRs and their cognate antigens interact at the initial site of contact.
• Changes in the submembrane network allow the formation of microclusters of 50 to 100 BCRs with associated coreceptors and signaling molecules.
• The B-cell membrane rapidly spreads over the target membrane, increasing the number of molecular interactions between the B cell and the antigen-bearing cell, peaking around 2 minutes after antigen contact.
• After maximal spreading, the area of contact between the cell and the artificial lipid membrane begins to contract, and by approximately 10 minutes after antigen contact, the antigen-receptor complex is gathered into a central, defined cluster.
• B cells in which the spreading response is deficient accumulate lower concentrations of antigen from their presenting cells.

BCR Complex Movement

• The BCR complex moves transiently into lipid rafts, which are highly ordered, detergent-insoluble, sphingolipid- and cholesterol-rich regions.
• Association of the BCR with lipid rafts brings the immunoreceptor tyrosine-based activation motifs (ITAMs) of the Igα and Igβ components of the BCR into close apposition with the raft-tethered, Src-family member tyrosine kinase, Lyn, and allows for initiation of the BCR signaling cascade.
• The BCR microclusters collapse into a single central cluster of receptors, called the central supramolecular activation cluster, or cSMAC, surrounded by a ring of adhesion molecules, including the integrin LFA-1, which is referred to as the peripheral supramolecular activation cluster, or pSMAC.
• The pSMAC is encircled by an actin ring forming the distal, or dSMAC.
• The integrins promote adhesion of the B cells to the antigen-presenting cells, lowering the threshold of antigen-binding affinity required for B-cell activation.
• This arrangement corresponds to that formed on T cells following recognition of antigen-presenting cells and is known as an "immunological synapse."

Key Concepts

• When the BCR recognizes its cognate antigen, the receptors on the B-cell membrane briefly spread over the surface of the antigen-presenting cell membrane, and then contract, resulting in B-cell receptor clustering.
• Receptor clustering leads to the formation of an immunological synapse between the B cell and its antigen.
• The B-cell synapse is made up of three concentric rings.
• Antigen-BCR complexes are clustered at the center, forming the cSMAC.
• A peripheral supramolecular activation cluster (pSMAC) is made up of adhesion molecules such as LFA-1.
• Polymerized actin forms the distal or dSMAC.

Antigen Binding to the BCR

• Antigen stimulation leads to the activation of transcription factors including NF-κB, NFAT, Egr-1, and Elk-1, which alter the cell’s transcriptional program.
• Molecular pathways trigger changes in membrane motility, in the expression of adhesion molecules and chemokine receptors, and in the production of anti-apoptotic molecules.

BCR Signal Transduction Pathways

• Antigen-mediated receptor clustering into the lipid raft regions of the membrane leads to Lyn-mediated phosphorylation of the signal transduction mediators Igα/β and the coreceptor CD19, and to the recruitment of the Syk and Tec family kinases, Syk and Btk.
• Syk and Btk are activated by both trans- and autophosphorylation steps.
• Phosphorylation of the adapter proteins BCAP (and BLNK) by Syk allows recruitment of phosphatidylinositol 3-kinase (PI3 kinase) to the membrane with generation of phosphatidylinositol trisphosphate (PIP3), which allows membrane localization of PDK1 and Akt and activation of Akt.
• Akt phosphorylates and inactivates the proapoptotic molecules Bax and Bad.
• Activation of the B-cell isoform of PLC, PLCγ2, occurs on binding to the phosphorylated adapter molecule BLNK and phosphorylation of PLCγ2 by Syk.
• PLCγ2 breaks down phosphatidylinositol bisphosphate (PIP2) to form diacylglycerol (DAG) and inositol trisphosphate (IP3).
• IP3 releases Ca2+ from intracellular stores with resultant activation of the NFAT pathway.
• DAG interacts with PKC as well as with the GTP exchange factor RasGRP, leading to activation of MAP kinase pathways, resulting in increases in Elk-1– and Egr-1–mediated transcription as well as CREB- and Jun-directed transcription.
• Cytoskeletal reorganization is mediated via Vav, activated on binding to the adapter protein BLNK.

Protein Tyrosine Kinase Families

• Members of the Src, Syk, and Tec families are implicated in the early steps of B-cell activation.
• Following antigen-induced clustering of the receptors into lipid rafts, raft-associated phosphatases begin the activation of the BCR-associated Src family kinases, Lyn and Fyn, which then autophosphorylate.
• They then go on to phosphorylate the ITAM-motif tyrosine residues on the Igα/β receptor–associated molecules.
• This provides attachment sites for the SH2 motifs of the adapter protein BLNK and for the kinase Syk.
• After binding to the Igα/β ITAMs, Syk is also activated by autophosphorylation and in turn phosphorylates the adapter protein BLNK, creating docking sites for multiple downstream components of the signaling pathway, including Bruton’s tyrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2).
• Btk and PLCγ2 are then themselves phosphorylated and activated.

Signalsome

• The "signalsome" refers to the signal-transducing molecular complex formed in activated B cells.
• It contains the BCR, Src family kinases Lyn, Fyn, and Blk, Syk, Btk, CD19, the adapter proteins BLNK and BCAP, and the signaling enzymes PLCγ2 and PI3 kinase.
• From this complex, further signals activate multiple cascades that signal changes in gene expression, cytoskeletal organization, and metabolism.
• Signal transduction pathways initiated in the BCR signalsome interact closely with other signaling pathways activated through cytokine receptors (IL-4 and IL-21), coreceptors (CD40), and survival factor receptors (BAFF-R).
• These pathways interface with negative signals from cell-surface molecules such as CD22 and CD32.

Activation Conclusion

• Antigen binding at the BCR leads to multiple changes in transcriptional activity, as well as in the localization and motility of B cells, which result in their enhanced survival, proliferation, differentiation, and eventual antibody secretion.

Key Concepts

• Antigen engagement by the BCR induces phosphorylation of tyrosine residues in the ITAMs of Igα and Igβ by Src family kinases.
• This phosphorylation initiates a response leading to the formation of a cytoplasmic signal-transducing complex called a signalsome.
• Multiple outcomes are possible following BCR antigen binding, depending on the strength and duration of antigen binding and are further regulated by interactions between the BCR and other cell receptors, including CD21, CD40, IL-4R, IL-21R, and BAFF-R.

B Cells and Coreceptors

• The immunoglobulin receptor on the B-cell membrane is noncovalently associated with three transmembrane molecules: CD19, CD21, and CD81.
• Antigens are sometimes presented to the BCR already covalently bound to complement proteins, in particular to the complement component C3d.
• The B-cell coreceptor CD21, otherwise known as CR2, specifically binds to C3d on C3d-coated antigens.
• This coengagement of the BCR and CD21 brings the coreceptor and the BCR into close apposition with one another.
• When this happens, tyrosine residues on the cytoplasmic face of the CD19 coreceptor become phosphorylated by the activated Src family kinases, providing sites of attachment for PI3 kinase.
• Localization of PI3 kinase to the coreceptor enhances cell survival and also results in alterations in the transcriptional program.
• CD19-mediated signaling is also vital in the membrane-spreading response.

Binding Conclusion

• The coreceptor CD21 can bind C3d complement fragments that are covalently associated with antigen, bringing the BCR into close contact with its coreceptor and enhancing antigen signaling.

Antigen-Presenting Cells Transfer

• There are two nonexclusive mechanisms by which this occurs.
• When a B cell forms a synapse with an antigen-presenting cell, the B cell polarizes the microtubule-organizing center toward the antigen contact site and lysosomes move toward the immunological synapse.
• On reaching the plasma membrane, the lysosomes spill their contents into the synaptic junction, acidifying the junction and allowing their proteolytic enzymes to cleave the bonds between the antigen and the antigen-presenting cell.
• The BCR continues to associate with downstream signaling molecules during the process of endocytosis, and efficient regulation of B-cell signaling is dependent on internalization of the receptor molecule.

Actomyosin Fibers

• Actomyosin fibers within the B cell exert force on the BCR, pulling on the antigen located on the antigen-presenting cell.
• If the affinity of the interaction between the BCR and the antigen is sufficiently high, the antigen is pulled out of the antigen-presenting cell membrane and into membrane invaginations of the B cell.
• From there, BCR-antigen complexes enter the endosomal system and are processed for antigen presentation.

Key Concept

• Antigen to be endocytosed by B cells may either be cleaved from the surface of the antigen-presenting cell by B cell–derived lysosomal proteases or “tugged” from the surface by binding to the BCR.

Internalization and Antigen Presentation

• Following antigen binding, most antigen-occupied BCR molecules are internalized, leaving just a few copies of the receptor on the cell surface.
• The endocytic vesicles carrying the BCR-antigen complex fuse with a specialized vesicle in which antigen-derived peptides, generated by endosomal proteases, are loaded onto the MHC class II molecules.
• The peptide-loaded MHC class II molecules are transported back to the B-cell surface for presentation to T cells.
• B-cell antigen processing and presentation results in the expression of peptide-loaded MHC class II molecules on the B-cell surface.
• BCR-mediated signaling causes increased expression of the costimulatory molecules CD80, CD86, and CD40 on the B-cell surface, preparing them for interactions with T cells.

B-Cell Communication

• A B cell that has taken up its specific antigen via BCR-mediated endocytosis is about 10,000-fold more efficient in presenting antigen to cognate T cells than is a non–antigen-specific B cell.
• Only B cells that can make antibody to that antigen present high levels of antigen to T cells.

Key Concepts

• BCR-mediated endocytosis transports antigen into vesicles, where it is broken down into peptides that are loaded onto MHC class II molecules and returned to the B-cell surface.
• Antigen engagement results in up-regulation of the expression of CD40, CD80, and CD86 on the B-cell surface.

B-Cell Migration

• A B cell needs to find and bind to a T cell specific for the same antigen.
• Lymph nodes can be brought outside the bodies of anesthetized animals, and the circulation of fluorescently tagged T, B, and antigen-presenting cells through these nodes can be visualized in real time.
• B cells are highly motile cells, programmed to respond to chemoattractant signals.

B-Cell Chemokine Receptor Migration

• The chemokine CXCL13 is made by follicular stromal cells and binds to heparan sulfate on stromal cells and collagen fibers in the follicle.
• When a B cell enters the lymph node, the B-cell chemokine receptor, CXCR5, responds to CXCL13 signaling and migrates to the follicle.
• Within the follicle, B cells move around, contacting follicular dendritic cells (FDCs) and other stromal cells to receive survival signals.
• As B cells move through the follicle, they may come into contact with antigen presented by subcapsular sinus macrophages (SCSMs) and dendritic cells or with soluble or cell-bound antigen that enters the follicle through the high endothelial venules.
• When the B cell meets an antigen capable of binding to its BCR, its previously random walk through the follicles begins to acquire a recognizable pattern, organized by successive modulations in chemokine receptors.
• The initial set of chemokine-directed migrations occurs in the first 1 to 3 hours after antigen contact.
• A few hours after antigen encounter, B cells up-regulate CCR7, whose ligands, CCL19 and CCL21, are secreted by stromal cells in the T-cell zones.

EBI2 Migration

• EBI2 receptor, whose ligand, 7α,25-dihydroxycholesterol, accumulates in the outer and interfollicular regions, directs the B cell to the outer regions of the follicle.
• This migration may allow the B cell access to any additional antigens that have accumulated in association with the subcapsular sinus macrophages.

B-Cell Activation

• By this time, the B cell has internalized and processed its antigen and displays antigenic peptides on its cell surface for presentation to T cells.
• Interactions between antigen-activated T and B cells continue to occur throughout this stage, with conjugate pairs of T cells readily observable during intravital microscopy experiments.
• The T-cell receptor reorients toward the synapse with the B cell, and the T cell begins to secrete interleukins, such as IL-4 and IL-21, that enable the B cell’s differentiation program to progress.
• Activated B cells up-regulate the expression of receptors for those cytokines.
• The receptor for IL-4 can be detected as early as 6 hours after antigen contact and reaches maximal levels of cell-surface expression at 72 hours.
• Interaction between CD40L (CD154) on T cells and CD40 on B cells is key to continued B-cell proliferation and differentiation.
• Changes in the expression of MHC class II antigens and of the two costimulatory molecules, CD80 and CD86, can be observed at 2 to 3 days post-stimulation

Cellular Migrations

• Antigen-specific B cells express GFP, and are therefore labeled green; antigen-specific T cells are labeled red; and the B-cell follicles are stained blue.
• T cells are located in the T-cell zones, and B cells are scattered throughout the follicles.
• With time postimmunization, B and T cells migrate into the boundaries of the T- and B-cell zones and into the interfollicular regions.
• After this period of intense communication with antigen-responsive T cells, at around 4 days postimmunization, some activated B cells down-regulate CCR7 and EBI2, enter the interior regions of the B-cell follicle, and begin the establishment of germinal centers.
• Other activated B cells within the same node retain EBI2 expression while decreasing CXCR5 expression and up-regulating the levels of cell-surface CXCR4 and move toward forming primary foci of proliferating B cells.
• These B cells will rapidly differentiate into plasmablasts and secrete antibodies.
• Some memory cells are generated very early in a primary immune response that express mainly IgM.
• Experiments suggest that an individual B cell is capable of generating daughter cells that have differentiated into antibody-producing plasma cells, GC-independent memory cells, and germinal center B cells.

Key Concepts

• Chemokine interactions with their receptors on B cells direct B-cell migration through the lymph node.
• T cells interact with their cognate B cells by binding to the processed antigen with their T-cell receptor (TCR), as well as by interactions between T-cell CD28 with B-cell CD80 and CD86, and between T-cell CD40L and B-cell CD40.
• Individual B cells may differentiate into plasma cells, early memory cells, or cells that enter the germinal center.

Transcription Factor Regulation

• Transcription factors that control whether antigen-stimulated B cells differentiate along the plasma cell or germinal center route are linked in a mutually regulatory network.
• Pax-5 and Bcl-6, along with low levels of IRF-4, favor the generation of proliferating, germinal center cells.
• BLIMP-1 and high levels of IRF-4 support the generation of antibody-secreting plasma cells.
• Other transcription factors, including IRF-8 and Bach2, also play modulating roles.
• Bcl-6 protein inhibits DNA repair, thus facilitating somatic hypermutation.
• BLIMP-1 supports the alternative splicing of Ig mRNA that generates the secreted form of Ig and represses MHC class II expression on the B-cell surface.
• Bcl-6 protein actively represses BLIMP-1 expression, which is also indirectly suppressed by Pax-5.
• Low levels of IRF-4 activate expression of the AID enzyme, which is critical for both SHM and CSR.

Key Concepts

• Following stimulation of primary B cells at the T-cell/B-cell border within the lymph node, some B cells differentiate quickly into plasma cells that form primary foci and secrete an initial wave of IgM antibodies.
• Other B cells from the antigen-stimulated clones migrate to the primary follicles and form germinal centers.

B-Cell Responses: Differentiation and Memory Generation

• Following activation by antigen in the presence of T cells, B cells can differentiate into plasma cells, memory cells, or activated germinal center B cells which secrete the high-affinity antibodies of the late primary response.
• Those B cells which became activated by an encounter with an antigen will enter the follicles, begin to divide rapidly, and get ready for further differentiation.

Primary Focus

• As described earlier, following antigen encounter, the first B cells to produce antibodies are the antibody-forming cells (AFCs) in the extrafollicular primary foci of lymph nodes and spleen.
• Plasmablasts formed on B-cell stimulation move to the medullary cords in the lymph nodes or to the border between the white and red pulp in the spleen, where they complete their differentiation to IgM-producing plasma cells.

Gene Coordination

• The process that starts with a naïve B cell and culminates in the formation of a mature plasma cell requires the coordinated expression and repression of hundreds of genes.
• Plasma cells depend for their growth and survival on the cytokines IL-6 and APRIL, which are produced by dendritic cells and monocytes within the lymph node.
• The AFCs initially produce only IgM antibodies, entry into the follicle is not necessary for the process of class switch recombination (CSR).
• However, neither IgM, nor IgG antibodies from the AFC, display evidence of somatic hypermutation (SHM), demonstrating conversely that the process of SHM occurs only in the germinal center.
• The size of extrafollicular AFC foci peaks around days 7 to 8 after antigen encounter, after which the foci decline in size; they are barely detectable by day 14and die by apoptosis.
• Plasma cells of the primary focus secrete large quantities of nonmutated IgM and IgG antibodies that provide early, protective humoral immunity and help to drive affinity maturation.

Germinal Centers

• Stimulated B cells entered the follicles following an encounter with antigen begin to divide rapidly and undergo further differentiation, resulting in the formation of specialized structures.
• GC's consist primarily of rapidly dividing B cells, they also contain follicular dendritic cells (FDCs), T follicular helper (TFH) cells, and macrophages.
• Depending on the nature of the antigen, the size of the GC peaks around 7 to 12 days after antigen stimulation, and GCs normally resolve within 3 to 4 weeks.

Darwinian Microcosm

• In the germinal centers, B cells undergo a period of intense proliferation, and their Ig genes are subjected to some of the most extraordinary processes in biology.
• First, the Ig variable region genes undergo extremely high rates of mutation, up to a thousand base pairs per generation. Then they express the BCR's within the GC.
• Somatic hypermutation and antigen selection, results in an increase in the affinity of secreted antibodies for the immunizing antigen
• B cells bearing receptors with higher affinity were selected for fur