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DYNAMIC INTERACTIONS AT THE BCR SYNAPSE
Just as TCRs form immunological synapses during contact with specific peptide–MHC, B‐cell receptors have also been found to exhibit similar behavior, particularly when antigen is presented on a membrane surface. Although B‐cells can be stimulated by soluble antigen, it is now widely accepted that the primary form of antigen that triggers B‐cell activation in vivo is localized to membrane surfaces. The most likely culprits here are the follicular DCs that are resident within lymph nodes, as well as macrophages and DCs that migrate there bearing gifts of antigen. Antigens can be immobilized on cell surfaces by complement or Fc receptors as immunocomplexes, or through direct binding to various scavenger receptors. An encounter between a B‐cell and membrane‐associated antigen provides the opportunity for the B‐cell membrane to spread along the opposing membrane, gathering sufficient antigen to trigger B‐cell activation, as well as providing an opportunity for other contacts to be made, such as those that can be provided by membrane integrins. This spreading response is driven by BCR engagement of antigen at the leading edge of the B‐cell and, apart from increasing the number of BCR–antigen contacts that are then available to trigger B‐cell activation, the spreading response also increases the amount of antigen that is ultimately concentrated and internalized by the B‐cell, leading to more efficient antigen presentation to activated T‐cells when the B‐cell subsequently goes looking for T‐cell help (Figure 7.30).
THE NATURE OF B‐CELL ACTIVATION
Similar to T‐cells, active or resting B‐cells are nondividing and activation through the BCR drives these cells into the cell cycle. As is the case for the TCR, the BCR (surface Ig) does not possess any intrinsic enzymatic activity. Once again, it is the accessory molecules associated with the antigen receptor that propagate activation signals into the B‐cell. It was noted in Chapter 4 that the BCR complex is composed of membrane‐anchored immunoglobulin that is associated with a disulfide‐linked Ig‐α and Ig‐β heterodimer, the cytoplasmic tails of which each contain a single ITAM motif (see Figure 4.4). As we will now discuss in more detail, antigen‐driven cross‐linking of the BCR results in the initiation of a (Bruton’s tyrosine kinase). Active Lyn also phosphorylates residues on CD19, a component of the B‐cell co‐receptor complex (discussed in detail in the next section) that rein forces signals initiated by the BCR (Figure 7.28). Syk fulfills a critical role within the B‐cell activation process; disruption of the gene encoding Syk in the mouse has profound effects on downstream events in B‐cell signaling and results in defective B‐cell development. In this respect, Syk serves a similar role in B‐cells to that served by ZAP‐70 inT‐cells. Active Syk phosphorylates and recruits BLNK (B‐cell linker; also called SLP‐65, BASH, and BCA) to the BCR complex. Upon phosphorylation by Syk, BLNK provides binding sites for phospholipase Cγ2 (PLCγ2), Btk, and Vav. Recruitment of Btk in close proximity to PLCγ2 enables Btk to phosphorylate the latter and increase its activity. Just as in the T‐cell signaling pathway, activated PLCγ2 initiates a pathway that involves hydrolysis of PIP2 to generate diacylglycerol and inositol trisphosphate and results in increases in intracellular calcium and PKC activation (Figure 7.28). PKC activation, in turn, results in activation of the NFκB and JNK transcription factors and increased intracellular calcium results in NFAT activation, just as it does in T‐cells.
Figure 7.27 Receptor cross‐linking recruits the BCR to lipid rafts. Antigen‐induced receptor cross‐linking recruits the BCR, which is normally excluded from membrane cholesterol‐rich lipid raft domains, to membrane lipid rafts where signaling proteins such as the protein tyrosine kinase Lyn reside. Stable recruitment of the BCR to rafts facilitates Lyn‐mediated phosphorylation of ITAMs within the cytoplasmic tails of the Ig‐α and Ig‐β accessory molecules that initiate the BCR‐driven signaling cascade.
The Vav family of guanine nucleotide exchange factors consists of at least three isoforms (Vav‐1, ‐2, and ‐3) and is known to play a crucial role in B‐cell signaling through activation of Rac1 and regulating cytoskeletal changes after BCR cross‐linking; Vav‐1‐deficient B‐cells are defective in proliferation associated with cross‐linking of the BCR (Figure 7.27).
Figure 7.28 Signaling cascade downstream of antigen‐driven Bcell receptor (BCR) ligation. Upon interaction with antigen, the BCR is recruited to lipid rafts where ITAMs within the Ig‐α/βheterodimer become phosphorylated by Lyn. This is followed by recruitment and activation of the Syk and Btk kinases. Phosphorylation of the B‐cell adaptor protein BLNK creates binding sites for several other proteins, including PLCγ2 that promotes PIP2 hydrolysis and instigates a chain of signaling events culminating in activation of the NFAT and NFkB transcription factors. The CD19 co‐receptor molecule is also phosphorylated by Lyn and can suppress the inhibitory effects of GSK3 on NFAT through the PI3K/Akt pathway. BCR stimulation also results in rearrangement of the cell cytoskeleton via activation of Vav that acts as a guanine nucleotide exchange factor for small G‐proteins such as Rac and Rho.
The BCR cross‐linking model seems appropriate for an understanding of stimulation by type 2 thymus‐independent antigens, as their repeating determinants ensure strong binding to, and cross‐linking of, multiple Ig receptors on the B‐cell sur face to form aggregates that persist owing to the long half‐life of the antigen and sustain the high intracellular calcium needed for activation. On the other hand, type 1 T‐independent antigens, such as the T‐cell polyclonal activators, probably bypass the specific receptor and act directly on downstream molecules such as diacylglycerol and protein kinase C, as Ig‐α and Ig‐β are not phosphorylated.
B‐cells require co‐stimulation via the B‐cell co‐receptor complex for efficient activation
Similar to T‐cells, B‐cells also require two forms of co‐stimulation to mount efficient effector responses. One form of co‐ stimulation takes place at the point of initial encounter of the BCR with its cognate antigen and is provided by the B‐cell co‐receptor complex that is capable of engaging with molecules such as complement that may be present in close proximity to the specific antigen recognized by the BCR. 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 CD40 on the B‐cell. This form of co‐stimulation requires that the B‐cell has internalized antigen, followed by processing and presentation on MHC class II molecules to an appropriate T‐cell. If the B‐cell is displaying an MHC–peptide combination recognized by the T‐cell, the latter will be stimulated to produce cytokines (such as IL‐4) as well as provide co‐stimulation to the B‐cell in the form of CD40L. We will consider the nature of the co‐stimulatory signals provided by the B‐cell co‐ receptor complex here and deal with CD40L‐based co‐stimulation in a separate section below.
Figure 7.29 The B‐cell co‐receptor complex provides co‐stimulatory signals for B‐cell activation through recruitment of a number of signaling molecules, including phosphatidylinositol 3‐kinase and Vav, which can amplify signals initiated through the B‐cell receptor. On mature B‐cells, CD19 forms a tetrameric complex with three other proteins: CD21 (complement receptor type 2), CD81 (TAPA‐1), and CD225 (interferon‐induced transmembrane protein 1) (LEU13).
The mature B‐cell co‐receptor complex (Figure 7.29) is composed of four components: CD19, CD21 (complement receptor type 2, CR2), CD81 (TAPA‐1), and CD225 (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 a innate immune response (complement) to synergize with the BCR to productively activate B‐cells. Imagine a bacterium that has activated complement and has become coated with the products of complement activation, including C3d. If the same bacterium 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, which effectively means that the B‐cell now receives two signals simultaneously. Signal 1 comes via the BCR and signal 2 via the co‐receptor complex. So how does simultaneous engagement of the co‐receptor complex and the BCR lead to enhanced B‐cell activation?
Well, the answer is that we don’t know for sure, but it is clear that CD19 plays an especially important role in this process. CD19 is a B‐cell‐specific transmembrane protein that is expressed from the pro‐B‐cell to the plasma‐cell stage and possesses a relatively long cytoplasmic tail containing nine tyrosine residues. Upon B‐cell receptor stimulation, the cytoplasmic tail of CD19 undergoes phosphorylation at several of these tyrosine residues (by kinases associated with the BCR) that creates binding sites on CD19 for several proteins, including the tyrosine kinase Lyn, Vav, and phosphatidylinositol 3‐kinase (PI3K). CD19 plays a role as a platform for recruitment of several proteins to the BCR complex (Figure 7.28), much in the same way that LAT functions in TCR activation.
Vav is recruited to CD19 upon phosphorylation of the latter by Lyn and, along with PI3K that is also recruited to CD19 as a result of Lyn‐mediated phosphorylation (Figure 7.28), plays a role in the activation of the serine/threonine kinase Akt; the latter may also enhance NFAT activation through neutralizing the inhibitory effects of GSK3 (glycogen synthase kinase 3) on NFAT. Because GSK3 can also phosphorylate and destabilize Myc and cyclin D, which are essential for cell cycle entry, Akt activation also has positive effects on proliferation of activated B‐cells.
Similar to the role that CD28 plays on T‐cells, the B‐cell co‐receptor amplifies signals transmitted through the BCR approximately 100‐fold. As we have discussed above, because CD19 and CR2 (CD21) molecules enjoy mutual association, this can be brought about by bridging the Ig and CR2 receptors on the B‐cell surface by antigen–C3d complexes bound to the surface of APCs. Thus, antigen‐induced clustering of the B‐cell co‐receptor complex with the BCR lowers the threshold for B‐cell activation by bringing kinases that are associated with the BCR into close proximity with the co‐receptor complex. The action of these kinases on the co‐receptor complex engages signaling pathways that reinforce signals originating from the BCR.
Figure 7.30 CD40–CD40L‐dependent B‐cell co‐stimulation by a T‐helper cell. Independently activated T‐ and B‐cells can interact if the Bcell is presenting the correct peptide–MHC complex sufficient for stimulation of the T‐cell. Successful antigen presentation by a B‐cell to an activated T‐helper cell results in CD40L‐dependent co‐stimulation of the B‐cell as well as the provision of cytokines, such as IL‐4, by the T‐cell that are essential for class switching, clonal expansion and differentiation to effector cells.
B‐cells also require co‐stimulation from T‐helper cells
Just as T‐cells require co‐stimulatory signals from DCs in the form of B7 ligands for productive activation (Figure 7.3), T‐dependent B‐cells also require co‐stimulation from T‐helper cells in order to cross the threshold required for clonal expansion and differentiation to effector cells (Figure 7.24). The sequence of events goes much like this. Upon encountering cognate antigen through direct binding to a microorganism, the BCR undergoes the initial activation events described above. This culminates in the internalization of the BCR, along with captured antigen, which is then processed and presented on MHC class II molecules (Figure 7.30). To continue the process of maturation to either a plasma cell or a memory cell, the B‐cell must now encounter a T‐cell capable of recognizing one of the antigenic peptides the B‐cell is now presenting from the antigen it has internalized. Note that this need not be the same epitope recognized by the B‐cell to undergo initial activation. Upon encountering a T‐cell with the appropriate TCR, the B‐cell provides stimulation to the T‐cell in the form of MHC–peptide as well as co‐stimulatory B7 signals (Figure 7.30). In turn, the T‐cell upregulates CD40 ligand (CD40L) that can provide essential co‐stimulation to the B‐cell, enabling the latter to become fully activated and undergo clonal expansion and class switching. If CD40L help is not forthcoming, B‐cells rapidly undergo apoptosis and are eliminated. This help is provided by a special class of T‐cell called a follicular helper T‐cell, a distinct branch of CD4+ T‐cells that express the cell surface receptor CXCR5, which targets them to B‐cell follicles in the secondary lymphoid organs. Thus, B‐cells and T‐cells provide mutual co‐stimulation as a means of reinforcing their initial activation signals (Figure 7.30). In effect, the B‐lymphocyte is acting as an APC and, as mentioned above, it is very efficient because of its ability to concentrate the antigen by focusing onto its surface Ig. Nonetheless, although a preactivated T‐helper can mutually interact with and stimulate a resting B‐cell, a resting T‐cell can only be triggered by a B‐cell that has acquired the B7 co‐stimulator and this is only present on activated, not resting, B‐cells.
Presumably the immune complexes on follicular DCs in germinal centers of secondary follicles can be taken up by the B‐cells for presentation to T‐helpers, but, additionally, the complexes could cross‐link the sIg of the B‐cell blasts and drive their proliferation in a T‐independent manner. This would be enhanced by the presence of C3 in the complexes as the B‐cell complement receptor (CR2) is co‐mitogenic.
Damping down B‐cell activation
We have already discussed how T‐cell enthusiasm for antigen can be dissipated by engaging CTLA‐4; similar mechanisms also operate to damp down signals routed through the BCR. Several cell surface receptors, including FcγRIIB, CD22, and PIRB (paired immunoglobulin‐like receptor B), have been implicated in antagonizing B‐cell activation through recruitment of the protein tyrosine phosphatase SHP‐1 to ITIMs (immunoreceptor tyrosine‐based inhibitory motifs) in their cytoplasmic tails. SHP‐1 impairs BCR signaling by antagonizing the effects of the Lyn kinase on Syk and Btk; by dephosphorylating both of these proteins SHP‐1 blocks recruitment of PLCγ2 to the BCR complex. Co‐ligation of the BCR with any of these receptors is therefore likely to block B‐cell activation. CD22 appears to be constitutively associated with the BCR in resting B‐cells and in this way may raise the threshold for B‐cell activation. Successful formation of a B‐cell receptor synapse ay physically exclude CD22 from the BCR complex.