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THE NATURE OF B‐CELL ACTIVATION

THE NATURE OF B‐CELL ACTIVATION

Similar to Tcells, active or resting Bcells 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 Bcell. It was noted in Chapter 4 that the BCR complex is composed of membraneanchored immunoglobulin that is associated with a disulfidelinked Igα and Igβ heterodimer, the cytoplasmic tails of which each contain a single ITAM motif (see Figure 4.4). As we will now discuss in more detail, antigendriven crosslinking of the BCR results in the initiation of a (Bruton’s tyrosine kinase). Active Lyn also phosphorylates residues on CD19, a component of the Bcell coreceptor 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 Bcell activation pro­cess; disruption of the gene encoding Syk in the mouse has profound effects on downstream events in Bcell signaling and results in defective Bcell development. In this respect, Syk serves a similar role in Bcells to that served by ZAP70 inTcells. Active Syk phosphorylates and recruits BLNK (Bcell linker; also called SLP65, 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 Tcell 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 Tcells.

Receptor cross‐linking recruits the BCR to lipid rafts

Figure 7.27 Receptor crosslinking recruits the BCR to lipid rafts. Antigeninduced receptor crosslinking recruits the BCR, which is normally excluded from membrane cholesterolrich 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 Lynmediated phosphorylation of ITAMs within the cytoplasmic tails of the Igα and Igβ accessory molecules that initiate the BCRdriven signaling cascade.

The Vav family of guanine nucleotide exchange factors consists of at least three isoforms (Vav1, 2, and 3) and is known to play a crucial role in Bcell signaling through activation of Rac1 and regulating cytoskeletal changes after BCR crosslinking; Vav1deficient Bcells are defective in proliferation associated with crosslinking of the BCR (Figure 7.27).

Signaling cascade downstream of antigen‐driven Bcell receptor (BCR) ligation

Figure 7.28 Signaling cascade downstream of antigendriven Bcell receptor (BCR) ligation. Upon interaction with antigen, the BCR is recruited to lipid rafts where ITAMs within the Igα/βheterodimer become phosphorylated by Lyn. This is followed by recruitment and activation of the Syk and Btk kinases. Phosphorylation of the Bcell 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 coreceptor 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 Gproteins such as Rac and Rho.


The BCR crosslinking model seems appropriate for an understanding of stimulation by type 2 thymusindependent antigens, as their repeating determinants ensure strong binding to, and crosslinking of, multiple Ig receptors on the Bcell sur­ face to form aggregates that persist owing to the long halflife of the antigen and sustain the high intracellular calcium needed for activation. On the other hand, type 1 Tindependent antigens, such as the Tcell polyclonal activators, probably bypass the specific receptor and act directly on downstream molecules such as diacylglycerol and protein kinase C, as Igα and Igβ are not phosphorylated.

 

Bcells require costimulation via the Bcell coreceptor complex for efficient activation

Similar to Tcells, Bcells also require two forms of costimulation 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 Bcell coreceptor 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 costimulation required by Bcells takes place after the initial encounter with antigen and is provided by Tcells in the form of membraneassociated CD40 ligand that engages with CD40 on the Bcell. This form of costimulation requires that the Bcell has internalized antigen, followed by processing and presentation on MHC class II molecules to an appropriate Tcell. If the Bcell is displaying an MHCpeptide combination recognized by the Tcell, the latter will be stimulated to produce cytokines (such as IL4) as well as provide costimulation to the Bcell in the form of CD40L. We will consider the nature of the costimulatory signals provided by the Bcell co receptor complex here and deal with CD40Lbased costimulation in a separate section below.

The B‐cell co‐receptor complex provides co‐stimulatory signals for B‐cell activation through recruitment of a number of signaling molecules

Figure 7.29 The Bcell coreceptor complex provides costimulatory signals for Bcell activation through recruitment of a number of signaling molecules, including phosphatidylinositol 3kinase and Vav, which can amplify signals initiated through the Bcell receptor. On mature Bcells, CD19 forms a tetrameric complex with three other proteins: CD21 (complement receptor type 2), CD81 (TAPA1), and CD225 (interferoninduced transmembrane protein 1) (LEU13).

The mature Bcell coreceptor complex (Figure 7.29) is composed of four components: CD19, CD21 (complement receptor type 2, CR2), CD81 (TAPA1), and CD225 (LEU13, interferoninduced transmembrane protein 1). CR2 is a receptor for the C3d breakdown product of complement and its presence within the BCR coreceptor complex enables a innate immune response (complement) to synergize with the BCR to productively activate Bcells. 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 Bcell, there is now an opportunity for CR2 within the BCR coreceptor complex to bind C3d, which effectively means that the Bcell now receives two signals simultaneously. Signal 1 comes via the BCR and signal 2 via the coreceptor complex. So how does simultaneous engagement of the coreceptor complex and the BCR lead to enhanced Bcell 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 Bcellspecific transmembrane protein that is expressed from the proBcell to the plasmacell stage and possesses a relatively long cytoplasmic tail containing nine tyrosine residues. Upon Bcell 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 3kinase (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 Lynmediated 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 Bcells.

Similar to the role that CD28 plays on Tcells, the Bcell coreceptor amplifies signals transmitted through the BCR approximately 100fold. 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 Bcell surface by antigen–C3d complexes bound to the surface of APCs. Thus, antigeninduced clustering of the Bcell coreceptor complex with the BCR lowers the threshold for Bcell activation by bringing kinases that are associated with the BCR into close proximity with the coreceptor complex. The action of these kinases on the coreceptor complex engages signaling pathways that reinforce signals originating from the BCR.

Figure 7.30 CD40–CD40Ldependent Bcell costimulation by a Thelper cell. Independently activated T and Bcells can interact if the Bcell is presenting the correct peptide–MHC complex sufficient for stimulation of the Tcell. Successful antigen presentation by a Bcell to an activated Thelper cell results in CD40Ldependent costimulation of the Bcell as well as the provision of cytokines, such as IL4, by the Tcell that are essential for class switching, clonal expansion and differentiation to effector cells.

Bcells also require costimulation from Thelper cells

Just as Tcells require costimulatory signals from DCs in the form of B7 ligands for productive activation (Figure 7.3), Tdependent Bcells also require costimulation from Thelper 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 Bcell must now encounter a Tcell capable of recognizing one of the antigenic peptides the Bcell is now presenting from the antigen it has internalized. Note that this need not be the same epitope recognized by the Bcell to undergo initial activation. Upon encountering a Tcell with the appropriate TCR, the Bcell provides stimulation to the Tcell in the form of MHC–peptide as well as costimulatory B7 signals (Figure 7.30). In turn, the Tcell upregulates CD40 ligand (CD40L) that can provide essential costimulation to the Bcell, enabling the latter to become fully activated and undergo clonal expansion and class switching. If CD40L help is not forthcoming, Bcells rapidly undergo apoptosis and are eliminated. This help is provided by a special class of Tcell called a follicular helper Tcell, a distinct branch of CD4+ Tcells that express the cell surface receptor CXCR5, which targets them to Bcell follicles in the secondary lymphoid organs. Thus, Bcells and Tcells provide mutual costimulation as a means of reinforcing their initial activation signals (Figure 7.30). In effect, the Blymphocyte 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 Thelper can mutually interact with and stimulate a resting Bcell, a resting Tcell can only be triggered by a Bcell that has acquired the B7 costimulator and this is only present on activated, not resting, Bcells.

Presumably the immune complexes on follicular DCs in germinal centers of secondary follicles can be taken up by the Bcells for presentation to Thelpers, but, additionally, the complexes could crosslink the sIg of the Bcell blasts and drive their proliferation in a Tindependent manner. This would be enhanced by the presence of C3 in the complexes as the Bcell complement receptor (CR2) is comitogenic.

Damping down Bcell activation

We have already discussed how Tcell enthusiasm for antigen can be dissipated by engaging CTLA4; similar mechanisms also operate to damp down signals routed through the BCR. Several cell surface receptors, including FcγRIIB, CD22, and PIRB (paired immunoglobulinlike receptor B), have been implicated in antagonizing Bcell activation through recruitment of the protein tyrosine phosphatase SHP1 to ITIMs (immunoreceptor tyrosinebased inhibitory motifs) in their cytoplasmic tails. SHP1 impairs BCR signaling by antagonizing the effects of the Lyn kinase on Syk and Btk; by dephosphorylating both of these proteins SHP1 blocks recruitment of PLCγ2 to the BCR complex. Coligation of the BCR with any of these receptors is therefore likely to block Bcell activation. CD22 appears to be constitutively associated with the BCR in resting Bcells and in this way may raise the threshold for Bcell activation. Successful formation of a Bcell receptor synapse ay physically exclude CD22 from the BCR complex.