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).
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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).
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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.
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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.
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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.
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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.
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Figure 4.6 The
B‐cell co‐receptor complex synergizes with the
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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.
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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.
