The B‐Cell Surface Receptor For Antigen (BCR)
The B‐cell displays a transmembrane immunoglobulin on its surface
In Chapter 2 we discussed the cunning system by which an antigen can be led inexorably to its doom by activating B‐cells that are capable of making antibodies complementary in shape to itself through interacting with a copy of the antibody molecule on the lymphocyte surface. It will be recalled that binding of antigen to membrane antibody can activate the B‐cell and cause it to proliferate, followed by maturation into a clone of plasma cells secreting antibody specific for the inciting antigen (Figure 4.1a).
Figure 4.1 B‐cells and T‐cells “see” antigen in fundamentally different ways. (a) In the case of B‐cells, membrane‐bound immunoglobulin serves as the B‐cell receptor (BCR) for antigen. (b) T‐cells use distinct antigen receptors, which are also expressed at the plasma membrane, but T‐cell receptors (TCRs) cannot recognize free antigen as immunoglobulin can. The majority of T‐cells can only recognize antigen when presented within the peptide‐binding groove of an MHC molecule. Productive stimulation of the BCR or TCR results in activation of the receptor‐bearing lymphocyte, followed by clonal expansion and differentiation to effector cells.
Immunofluorescence staining of live B‐cells with labeled anti‐immunoglobulin (anti‐Ig) (e.g., Figure 2.8c) reveals the earliest membrane Ig to be of the IgM class. Each individual B‐cell is committed to the production of just one antibody specificity and so transcribes its individual rearranged VJCk (or λ) and VDJCμ genes. Ig can be either secreted or displayed on the B‐cell surface through differential splicing of the pre‐mRNA transcript encoding a particular immunoglobulin. The initial nuclear μ chain RNA transcript includes sequences coding for hydrophobic transmembrane regions that enable the IgM to sit in the membrane where it acts as the BCR, but if these are spliced out, the antibody molecules can be secreted in a soluble form (Figure 4.2).
Figure 4.2 Splicing mechanism for the switch from the membrane to the secreted form of IgM. Alternative processing determines whether a secreted or membrane‐bound form of the μ heavy chain is produced. If transcription termination or cleavage occurs in the intron between Cμ4 and M1, the Cμ4 poly‐A addition signal (AAUAAA) is used and the secreted form is produced. If transcription continues through the membrane exons, then Cμ4 can be spliced to the M sequences, resulting in the M2 poly‐A addition signal being utilized. The hydrophobic sequence encoded by the exons M1 and M2 then anchors the receptor IgM to the membrane. For simplicity, the leader sequence has been omitted. = introns.
As the B‐cell matures, it coexpresses a BCR utilizing surface IgD of the same specificity. This surface IgM surface IgD B‐cell phenotype is abundant in the mantle zone lymphocytes of secondary lymphoid follicles (see Figure 6.15d) and is achieved by differential splicing of a single transcript containing VDJ, Cμ, and Cδ segments producing either membrane IgM or IgD (Figure 4.3). As the B‐cell matures further, other isotypes such as IgG may be utilized in the BCR.
Figure 4.3 Surface membrane IgM and IgD receptors of identical specificity appear on the same cell through differential splicing of the composite primary RNA transcript. Leader sequences again omitted for simplicity.
Surface immunoglobulin is complexed with associated membrane proteins
Because secreted immunoglobulin is no longer physically connected to the B‐cell that generated it, there is no way for the B‐cell to know when the secreted Ig has found its target antigen. In the case of membrane‐anchored immunoglobulin however, there is a direct link between antibody and the cell making it and this can be exploited to instruct the B‐cell to scale‐up production. As any budding industrialist knows, one way of increasing production is to open up more manufacturing plants, and another is to increase the rate of productivity in each one. When faced with the prospect of a sudden increase in demand for their particular product, B‐cells do both of these things, through clonal expansion and differentiation to plasma cells. So how does the BCR spur the B‐cell into action upon encounter with antigen?
Unlike many plasma membrane receptors that boast all manner of signaling motifs within their cytoplasmic tails, the corresponding tail region of a membrane‐anchored IgM is a mis erable three amino acids long. In no way could this accommodate the structural motifs required for interaction with the adaptor proteins, intracellular protein kinases, or phosphatases that typically initiate signal transduction cascades. With some difficulty, it should be said, it eventually proved possible to isolate a disulfide‐linked heterodimer, Ig‐α (CD79a) and Ig‐β (CD79b), which copurifies with membrane Ig and is responsible for transmitting signals from the BCR to the cell interior (Figure 4.4). Both Ig‐α and Ig‐β have an extracellular immunoglobulin‐type domain, but it is their C‐terminal cytoplasmic domains that are obligatory for signaling and which become phosphorylated upon cross‐linking of the BCR by antigen (Figure 4.5), an event also associated with rapid Ca2+ mobilization.
Figure 4.4 Model of B‐cell receptor (BCR) complex. The Ig‐α/Ig‐β heterodimer is encoded by the B‐cell‐specific genes mb‐1 and B29, respectively. Two of these heterodimers are shown with the Ig‐α associating with the membrane‐spanning region of the IgM μ chain. The Ig‐like extracellular domains are colored blue. Each tyrosine (Y)‐containing box possesses a sequence of general structure Tyr.X2.Leu.X7.Tyr.X2.Ile (where X is not a conserved residue), referred to as the immunoreceptor tyrosine‐based activation motif (ITAM). On activation of the B‐cell, these ITAM sequences act as signal transducers through their ability to associate with and be phosphorylated by a series of tyrosine kinases. Note that while a κ light chain is illustrated for the surface IgM, some B‐cells utilize a λ light chain.
Figure 4.5 B‐cell receptor clustering drives activation. Activation of the BCR complex through antigen engagement results in signal propagation as a consequence of phosphorylation of the intracellular ITAMs within the Ig‐α/Ig‐β
Ig‐α and Ig‐β each contain a single ITAM (immunoreceptor tyrosine‐based activation motif) within their cytoplasmic tails and this motif contains two precisely spaced tyrosine residues that are central to their signaling role (Figure 4.4 and Figure 4.5). Engagement of the BCR with antigen leads to rapid phosphorylation of the tyrosines within each ITAM, by kinases associated with the BCR, and this has the effect of creating binding sites for proteins that have an affinity for phosphorylated tyrosine residues. In this case, a protein kinase called Syk becomes associated with the phosphorylated Ig‐α/‐β heterodimer and is instrumental in coordinating events that culminate in entry of the activated B‐cell into the cell cycle to commence clonal expansion. We will revisit this topic in Chapter 7 where the details of the BCR signal transduction cascade will be elaborated upon in greater detail.
Specific antigen drives formation of B‐cell receptor microclusters
Recent studies suggest that many of the BCRs do not freely diffuse within the plasma membrane with their associated Ig‐α/β heterodimers, but are constrained within specific zones by the underlying actin cytoskeleton. The actin cytoskeleton does not make contact with the BCR directly but corrals the receptor into confinement zones through interaction with membrane ezrin. There is a good reason for this confinement, as this appears to be required to prevent spontaneous formation of BCR microclusters. These appear to be the structures that are capable of transmitting signals into the B‐cell that rep resents an activation stimulus. BCR microclusters are made up of 50–500 BCR molecules and have been visualized on the surface of B‐cells using advanced microscopy techniques. Indeed, mere depolymerization of the actin cytoskeleton appears to be sufficient to permit weak B‐cell activation signals to occur spontaneously, without any requirement for antigen, suggesting that cytoskeleton‐based confinement is necessary and acts as a “safety catch” on BCR triggering. Indeed, weak background or “tonic” BCR signals appear to be necessary for B‐cell development, as interference with this situation results in death of developing B‐cells. Presumably a small fraction of the BCR pool that is freely diffusible within the plasma membrane provides this tonic signaling.
B‐cell activation appears to require that many BCRs become dislodged from their confinement zones to become recruited into microclusters, an event that very recent evidence suggests is achieved through antigen‐induced conformational changes within the antibody constant region that permits self‐association within the membrane. More effective BCR stimulation is also achieved through cross‐linking of the BCR with its co‐receptor complex, which is discussed below. B‐cell activation through BCR stimulation alone is possible, but the former tends to lead to low‐affinity IgM production and is far less preferable to co‐stimulation via the BCR co‐receptor complex.
There is also a growing appreciation that while B‐cells can be stimulated by soluble antigen, the primary form of antigen that triggers B‐cell activation in vivo is predominantly localized to membrane surfaces. The most likely source of membrane‐localized antigen are the follicular dendritic cells that are resident within lymph nodes and are specialized at capturing complement‐decorated antigen complexes that diffuse into these lymphoid tissues. Interaction between a B‐cell and membrane‐immobilized antigen provides the opportunity for the B‐cell membrane to spread along the opposing antigen‐bearing membrane, gathering sufficient antigen to trigger B‐cell microcluster formation and activate the B‐cell.
In addition to providing an optimal activation stimulus, there might be another reason why B‐cells are keen to engage as many BCRs as possible with specific antigen. This is because activated B‐cells require help, in the form of cytokines and CD40 receptor stimulation, from T‐helper cells, to undergo class switching and somatic hypermutation. This help is only forthcoming if the B‐cell can present antigen to T‐cells in the context of MHC class II molecules. Thus, the more antigen captured by a stimulated B‐cell, the more efficient it will be in subsequently acquiring T‐cell help. Thus, spreading along an antigen‐coated surface facilitates engagement of many BCRs with antigen, which can then be internalized by the B‐cell to be processed and presented to T‐helper cells. We will revisit the issue of T‐cell–B‐cell interactions in Chapters 7 and 8 when we will look at these events in more detail.
The B‐cell co‐receptor complex synergizes with the BCR to activate B‐cells
We have already made reference to the two‐signal model for activation of naive T‐cells. Similarly, B‐cells also require two signals (with some exceptions) to become productively activated and this most likely represents a safeguard to limit the production of autoantibodies. Indeed, as we will discuss in more detail in Chapter 7, there are actually two distinct types of co‐stimulation a B‐cell needs to receive, at different times, for truly optimum activation and subsequent class switching and affinity maturation. One form of co‐stimulation takes place at the point of initial encounter of the BCR with its cog nate antigen and is provided by the B‐cell co‐receptor complex that is capable of engaging with molecules such as complement that may be decorating the same surface (e.g., on a bacterium) displaying the specific antigen recognized by the BCR (Figure 4.6). The other form of co‐stimulation required by B‐ cells takes place after the initial encounter with antigen and is provided by T‐cells in the form of membrane‐associated CD40 ligand that engages with surface CD40 on the B‐cell. We will discuss CD40L‐dependent co‐stimulation in Chapter 7, as this is not required for initial activation but is very important for class switching and somatic hypermutation.
Figure 4.6 The B‐cell co‐receptor complex synergizes with the
BCR to activate B‐cells. The B‐cell co‐receptor complex is composed of four components: CD19, CD21 (complement receptor type 2, CR2), CD81 (TAPA‐1), and CD225 (LEU13, interferon‐induced transmembrane protein 1, see also Figure 7.29). Because CR2 is a receptor for the C3d breakdown product of complement, its presence within the BCR co‐receptor complex enables complement to synergize with the BCR, thereby enhancing B‐cell activation signals.
The B‐cell co‐receptor complex (Figure 4.6) is composed of four components: CD19, CD21 (complement receptor type 2, CR2), CD81 (TAPA‐1), and LEU13 (interferon‐induced transmembrane protein 1). CR2 is a receptor for the C3d breakdown product of complement and its presence within the BCR co‐receptor complex enables complement to synergize with the BCR, thereby enhancing cross‐linking, which drives microcluster formation. Thus, in situations in which a bacterium has activated complement and is coated with the products of complement activation, when it is subsequently captured by the BCR on a B‐cell there is now an opportunity for CR2 within the BCR co‐receptor complex to bind C3d on the bacterium. This effectively means that the B‐cell now receives two signals simultaneously. Signal one comes via the BCR and signal two via the co‐receptor complex.