The B-lymphocyte antigen receptor is a transmembrane form of an antibody molecule associated with two signaling chains. We described the structure of antibodies in detail in Chapter 5. Here we will focus on some salient features of the membrane forms of Ig and their associated proteins and discuss how they deliver signals to B cells. Because the signaling pathways are much like those in T cells, we will summarize these without great detail. As noted earlier, there are both similarities and significant differences between B- and T-cell antigen receptors (see Table 1).
Table1. Properties of Lymphocyte Antigen Receptors: T-Cell Receptor (TCR) and Immunoglobulins (Igs)
Structure of the B-Cell Receptor for Antigen
Membrane IgM and IgD are the antigen receptors of naive B cells. They have short cytoplasmic tails consisting of only three amino acids (lysine, valine, and lysine), which are too small to transduce signals after the recognition of antigen. Ig-mediated signals are delivered by two other molecules called Igα and Igβ that are disulfide linked to one another and are expressed in B cells noncovalently associated with membrane Ig (Fig. 1). These proteins have functions in B cells that are similar to the functions of CD3 and ζ proteins in TCR signaling. They contain ITAM motifs in their cytoplasmic tails, are required for the transport of membrane Ig molecules to the cell surface, and together with membrane Ig form the BCR complex. The tails of Igα and Igβ are noncovalently associated with SRC family tyro sine kinases, including LYN, FYN, and BLK. BCR complexes in class-switched B cells, including memory B cells, contain mem brane Igs that may be of the IgG, IgA, or IgE classes. Membrane IgG and IgE molecules have longer cytoplasmic tails that contain a conserved aspartate (or glutamate)-tyrosine arginine-asparagine-methionine sequence called an Ig tail tyro sine (ITT) motif that is similar in sequence (and function) to a signaling motif in the cytoplasmic tail of the CD28 costimulatory receptor in T cells.
Fig1. B-cell antigen receptor complex. As seen in the left panel, membrane immunoglobulin M (IgM) (and IgD) on the surface of mature B cells is associated with the invariant Igβ and Igα molecules, which contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic tails that mediate signaling functions. In the right panel, the cytoplasmic tail of membrane IgG (and the same is true for membrane IgE) contains a tyrosine containing motif called the Ig tail tyrosine (ITT) motif that helps amplify B-cell receptor signaling in memory B cells.
Signal Initiation by the B-Cell Receptor
Signal initiation by antigens occurs by cross-linking of the BCR. Cross-linked Ig receptors enter lipid rafts, where many adaptor proteins and signaling molecules are concentrated, along with SRC family tyrosine kinases. Thus, cross-linking of membrane IgM and IgD by multivalent antigens brings molecules of Igα- and Igβ-associated SRC family kinases (like LYN, FYN or BLK) close to one another. The subsequent physical interaction of the kinase molecules leads to the phosphorylation of the tyrosine residues on the ITAMs of Igα and Igβ. The phosphorylation of ITAM tyrosine residues triggers all subsequent signaling events downstream of the BCR (Fig. 2). The phosphorylated tyro sine residues in the ITAMs of Igα and Igβ provide docking sites for the tandem SH2 domains of the SYK tyrosine kinase. SYK is homologous to ZAP70 and has similar functions in B cells as ZAP70 does in T cells. SYK is activated when it associates with phosphorylated tyrosines of ITAMs and may itself be phosphorylated on specific tyrosine residues by BCR-associated SRC family kinases, leading to further activation. Both SRC-family kinases and SYK contribute to the activation of BTK, an important tyro sine kinase in B cells. If the antigen is monovalent and incapable of cross-linking multiple Ig molecules, some signaling may nevertheless occur, but additional activation by helper T cells may be necessary to fully activate B cells.
Fig2. Signal transduction by the B-cell receptor complex. Antigen-induced cross-linking of membrane immunoglobulin (Ig) on B cells leads to clustering and activation of SRC family tyrosine kinases and tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs in the cytoplasmic tails of the Igα and Igβ molecules. This leads to docking of SYK and subsequent tyrosine phosphorylation events, as depicted. Several signaling cascades follow these events, as shown, leading to the activation of several transcription factors. These signal transduction pathways are similar to those described in T cells. ERK, Extracellular receptor–activated kinase; GDP, guanosine diphosphate; GTP, guanosine triphosphate; JNK, c-Jun N-terminal kinase; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-κB; PIP3, phosphatidylinositol trisphosphate; PKC, protein kinase C; PLCγ, phospholipase Cγ.
Role of the CR2/CD21 Complement Receptor as a Coreceptor for B Cells The activation of B cells is enhanced by signals that are provided by complement proteins and the CD21 coreceptor com plex, which link innate immunity to the adaptive humoral immune response (Fig. 3). Microbial surfaces and released polysaccharides can activate the complement system by the alternative = and lectin pathways in the absence of antibodies during innate immune responses. Proteins and other antigens that do not activate complement directly may be bound by preexisting antibodies or by antibodies produced early in the response, and these antigen-antibody complexes can activate complement by the classical pathway. Recall that complement activation results in the proteolytic cleavage of complement proteins. The key component of the system is a protein called C3, and its cleavage results in the production of a molecule called C3b that binds covalently to the microbe or antigen-antibody com plex. C3b is further degraded into a fragment called C3d, which remains bound to the microbial surface or the antigen-antibody complex. B lymphocytes express a receptor for C3d called the type 2 complement receptor (CR2, or CD21). The complex of C3d and antigen or C3d and antigen-antibody complex binds to B cells, with the membrane Ig recognizing antigen and CR2 recognizing the bound C3d (see Fig. 3). CR2 is expressed on mature B cells as a complex with two other membrane proteins, CD19 and CD81 (also called TAPA1). The CR2-CD19-CD81 complex is often called the B-cell coreceptor complex because CR2 binds to antigens through attached C3d at the same time that membrane Ig binds directly to the antigen. Binding of C3d to the B-cell complement receptor brings CD19 in proximity to BCR-associated kinases, and the cytoplasmic tail of CD19 rapidly becomes tyrosine phosphorylated. This leads to the activation of PI3kinase, which generates PIP3, which in turn binds and activates BTK and PLCγ2, in a manner analogous to PDK1 activation and ITK and PLCγ1 activation in T cells. The net result of coreceptor activation is that the response of the antigen-stimulated B cell is greatly enhanced.
Fig3. Role of complement in B-cell activation. B cells express a complex of the CR2 complement receptor, CD19, and CD81. Microbial antigens that have bound the complement fragment C3d can simultaneously engage both the CR2 molecule and the membrane immunoglobulin (Ig) on the surface of a B cell. This leads to the initiation of signaling cascades from both the B-cell receptor complex and the CR2 complex, because of which the response to C3d-antigen complexes is greatly enhanced compared with the response to antigen alone.
Complement proteins are not the only innate immune triggers of B cells. Other innate immune ligands, including flagellin and microbial nucleic acids, can also activate B cells by activating innate immune receptors in B cells, such as Toll-like receptors (TLRs). In rodents (though not in humans), lipolysaccharides can also activate B cells.
Signaling Pathways Downstream of the B-Cell Receptor
After antigen binding to the BCR, ITAM phosphorylation and the recruitment and activation of SYK adaptor proteins recruit specific enzymes and activate numerous downstream signaling pathways (see Fig. 3). Activated SYK phosphorylates critical tyrosine residues on adaptor proteins such as SLP65 (also called BLNK). This facilitates the recruitment to these adaptor proteins of other SH2 domain– and phosphotyrosine-binding (PTB) domain–containing enzymes, including guanine nucleotide exchange proteins that can separately activate many downstream signaling molecules including RAS, RAC, PLCγ2, and the BTK tyrosine kinase. Recruitment facilitates the activation of these downstream effectors, each generally contributing to the activation of a distinct signaling pathway.
• The RAS-MAP kinase pathway is activated in antigen stimulated B cells. The GTP/GDP exchange factor SOS is recruited to the adaptor protein SLP65 through the binding of GRB2; RAS is then converted by SOS from an inactive GDP-bound form to an active GTP-bound form. Activated RAS contributes to the activation of the ERK MAP kinase pathway, as discussed earlier for T-cell signaling. In a parallel fashion, the activation of the small G protein RAC may contribute to the activation of the JNK MAP kinase pathway.
• A phosphatidylinositol-specific phospholipase C (PLC) is activated in response to BCR signaling, and this in turn facilitates the activation of downstream signaling pathways. In B cells, the dominant isoform of PLC is the γ2 isoform, whereas T cells express the related γ1 isoform of the enzyme. PLCγ2 becomes active when it binds to the adaptor protein BLNK and is phosphorylated by BTK. As described in the context of TCR signaling, active PLC breaks down membrane PIP2 to yield soluble IP3 and leaves DAG in the plasma membrane. IP3 mobilizes calcium from intracellular stores, leading to a rapid elevation of the concentration of cytoplasmic calcium ions, which is subsequently augmented by an influx of calcium from the extracellular milieu. Calcium signaling con tributes to NFAT activation in B cells as well, analogous to events described in T cells. In the presence of elevated calcium, DAG activates some isoforms of PKC (mainly PKCβ in B cells), which phosphorylate downstream proteins on serine/threonine residues.
• PKCβ activation downstream of the BCR contributes to the activation of NF-κB in antigen-stimulated B cells. This process is similar to that in T cells triggered by PKCθ, the PKC isoform present in T cells.
• As described for T-cell activation, the phosphorylation of specific tyrosine-containing motifs on a number of adaptors in B cells allows the recruitment and activation of PI3-kinase. This enzyme facilitates critical cellular events, including cell survival, in activated B cells.
These signaling cascades ultimately lead to the activation of transcription factors that induce the expression of genes whose products are required for functional responses of B cells. Some of the transcription factors that are activated by antigen receptor–mediated signal transduction in B cells are FOS (downstream of RAS and ERK activation), JUNB (downstream of RAC and JNK activation), and NF-κB (downstream of BTK, PLCγ2, and PKCβ activation). We described these earlier in the context of T-cell signaling pathways. These and other transcription factors, many not mentioned here, are involved in stimulating proliferation and differentiation of B cells.
The same signaling pathways are used by membrane IgM and IgD on naive B cells and by IgG, IgA, and IgE on B cells that have undergone class (isotype) switching because all of these membrane Ig classes associate with Igα and Igβ. However, in memory B cells expressing membrane IgG or IgE, the tyrosine residue in the ITT motif is phosphorylated, recruits the GRB2 adaptor and enhances both ERK activation and Ca++ signaling.
