1. B Cell Receptor Signalling
Immunoglobulin molecule
CD79b (Igb)
CD79a (Iga)
The surface Ig molecule plus one molecule each of CD79a and CD79b together form the B cell receptor for antigen (BCR). The Ig molecule has a very short cytoplasmic tail and cannot signal by itself. CD79a and CD79b contain ITAMs and are the signal transducing parts of the receptor.

2. Structure of the BCR
CD79a and CD79b heterodimerise with each other. One heterodimer is found associated with one Ig molecule.
BCRs form small oligomeric groups on the surface of B cells.
On IgM+, IgD+ cells, the oligomers contain only IgM or IgD, there are no IgM/IgD oligomers.
The BCR has no intrinsic signalling activity. It acts as a scaffold to build a signalling system.
BCRs are not found in lipid rafts. They move into rafts following BCR cross-linking.

3. Signal Spreading Model
The B cell can respond to the level of BCR cross-linking i.e. the more cross-linking that occurs, the bigger the signal that is transduced.
The simple model proposed that the magnitude of the signal was related to the number of ligand-bound receptors in a linear manner. However, this model could not account for the sensitivity of the response at low ligand levels.
The signal spreading model proposes that ligand-bound receptors can cause ligand-independent activation of their neighbours. This would allow signals to be transduced even at very low levels of antigen binding. However, if signal spreading was constant at all ligand concentrations, it would simply shift the dose-response of the BCR. At high concentrations of ligand there is less signal spreading. This is achieved by negative feedback mechanisms whose activation requires higher concentrations of ligand.

4. * * * * * * * * * * * * * * * * Signal Spreading – How it Works
Pre-existing oligomer
Antigen cross-links a small number of BCRs which are then activated * * * *
The signal spreads to neighbouring unbound receptors which then become activated * * * * * * * * * * * *

5. Conventions in Signalling Diagrams
Symbol
Meaning
A inhibits B A B
A activates B, or A is converted to B A B
A binds to B A B

6. * * * * * * * BCR Signalling – the First Steps lyn
Following cross-linking, the BCR moves into lipid rafts.
The src family kinase, lyn, is targetted to lipid rafts.
When the BCR enters the raft, it encounters lyn. Lyn phosphorylates the ITAMs in CD79a and CD79b.
The tyrosine kinase, syk, is associated with CD79a and CD79b. When the ITAMs are phosphorylated, syk associates with them, becoming more active.
Syk is then phosphorylated by lyn, becoming even more active.
BCR Cross-linking * * * * * * *
lyn
BCR is recruited to rafts where it encounters lyn
Lyn phosphorylates ITAMs
Syk is recruited to phosphorylated ITAMs
Lyn phosphorylates syk
Lyn found in rafts

7. * * * * PIP3 PI3-K btk PIP5K rac vav syk CD79b CD79a lyn BL N K PLCg2
Lyn is responsible for initiating BCR signalling. Lyn phosphorylates PI3-kinase causing the production of PIP3. PIP3 targets the key adapter molecule, BLNK, to the plasma membrane. The tyrosine kinase btk is also targetted to the membrane where it is initially phosphorylated and activated by lyn. BLNK assembles a signalling complex leading to the activation of PLCg2 (and therefore a Ca2+ signal) and to the activation of vav. Once the complex is formed, syk takes over and phosphorylates and activates both btk and PI3-K, thus maintaining the signal.

8. * * * * * * * * * * * * * CD19 PIP3 PI3-K vav syk CD79b CD79a CD79b
CD19 is a transmembrane protein with a very large cytoplasmic domain. It is present on follicular dendritic cells and on B cells from the early pre-B stage until plasma cell differentiation. It is found associated with the BCR, or with CD21, or on its own in the plasma membrane.
The cytoplasmic domain has multiple tyrosines which are phosphorylated following BCR ligation. CD19 acts as a adaptor molecule and amplifies BCR signals. The major role of CD19 is to maintain PIP3 production.

9. PtdIns 3 kinase and Ca2+ syk lyn PLCg btk PtdIns(4,5)P2
PtdIns(3,4,5)P3 targets both PLCg and btk to the plasma membrane. Btk is then phosphorylated by lyn, autophosphorylated by itself, and finally phosphorylates and activates PLCg. After 10 to 20 seconds, syk takes over the role of lyn. The continued presence of PtdIns(3,4,5)P3 and the continued activity of syk are required to maintain a Ca2+ signal.
syk
Ca2+
Ins(1,4,5)P3
lyn
PLCg
btk
PKC
DAG
PtdIns(4,5)P2
PtdIns(3,4,5)P3
PtdIns 3 kinase

10. PtdIns 3 kinase and Proliferation
PtdIns(3,4)P2
PTEN
SHIP
PtdIns 3 kinase
PTEN
PtdIns(4)P
PtdIns(4,5)P2
PtdIns(3,4,5)P3
PtdIns 5 kinase
PtdIns 3 kinase
PKB (Akt)
PDK-1
Caspase 9
Bad
S6 kinase
GSK 3

11. Vav: effects on the cytoskeleton
Ca2+
Dissociates profilin and gelsolin from actin. Promotes interaction between actin and the cytoskeleton proteins vinculin, talin and zyxin.
Ins(1,4,5)P3
PKC
DAG
PtdIns
PtdIns(4)P
PtdIns(4,5)P2
PtdIns(3,4,5)P3
(Binds to vav via the PH domain on vav, thus targetting vav to the plasma membrane).
PtdIns 4 kinase
PtdIns(4)P 5 kinase
PtdIns 3 kinase
cdc42
rac1
RhoA
Vav

12. Vav: effects on MAPkinases
cdc42
rac1
RhoA
MEKK1
NB, this is not the major pathway of vav action.
Stimulated by PKCm and Ca2+. The point of action of these molecule on this kinase cascade is unknown.
MKK4 (Sek1)
p38
Jnk1 (SAPK)
MAPKAP kinase 2
homodimerisation
C-jun; atf-2; p53; ets
Enters nucleus

13. Ras Pathway Ras GAP ras sos raf1 PKCm MEK1 MEK2 ERK1 ERK2
homodimerisation
homodimerisation
nucleus
Fos; jun; ets

14. shc Shc regulates the ras pathway PIP3 PI3-K sos Gab1 ras ? Grb2 syk
Shc, Grb2 and Gab1 are adaptors. Syk phosphorylates shc. This allows shc to bind to Grb2 and Gab1 via their SH2 domains. Gab1 recruits shc to PIP3 thus bringing shc to the plasma membrane. Shc binds to Grb2 (which is bound to sos), therefore recruiting Grb2 and sos to the membrane. Sos is a guanine nucleotide exchange factor for ras. Ras is membrane-bound, therefore the net result of recruitment of shc to the plasma membrane is to bring sos into contact with its substrate – ras.

15. BCR Signalling in B Cell Development
anergy
Receptor editing
apoptosis
Pre-B cell receptor (Igm, l5, Vpre-B, CD79a, CD79b)
Pro-B cell receptor (calnexin, CD79a, CD79b)
autoreactive
Early pro-B
Late pro-B
Large pre-B
Small pre-B
Immature B
Signalling through the BCR results in different outcomes depending on the stage of differentiation.
Pro-BCR signalling may be required for transition to large pre-B stage. This is ligand-independent.
Pre-BCR signalling is required for transition to small pre-B stage. It is currently thought that this is ligand-independent.
BCR signalling on immature B cells leads to apoptosis or anergy. This is a mechanism for eliminating autoreactive B cells.
BCR signalling on mature B cells leads to proliferation.
Transitional B
Marginal zone B B1
Mature B

16. BCR Signalling in B Cell Development
Why does signalling through the same receptor result in different outcomes? The full picture is far from clear, but some differences have been noted:
The pre-BCR is located in lipid rafts; BCRs at other stages of B cell development are not. This may allow the pre-BCR to constitutively signal.
The BCR on immature B cells does not move to lipid rafts after cross-linking in contrast to the BCR on mature B cells.
The nature of the antigen appears to make a difference e.g. for immature B cells, soluble self antigen leads to anergy, while membrane-bound self antigen leads to apoptosis. This may relate to the degree of cross-linking that the antigen can induce.

17. The B Cell Synapse
Just like T cells, B cells can form a synapse with an antigen-presenting cell. See the following references for full details: Nature 411: and Nature Immunology 2:
The B cell synapse relies on the presentation of intact antigen by an antigen-presenting cell. The APC can present the antigen as an immune complex of antibody plus antigen (bound via Fcg receptors to the APC) or as complement-coated antigen (bound via CD21 or CD35 on the APC).
On the B cell membrane the synapse excludes negative regulators of BCR signalling, such as CD22 and CD45, from the BCR complex. This allows a strong signal to be transduced.
The role of integrins was not examined.

18. Modifying BCR Signals
B cells need to be able to respond to their environment. They need to have the capacity to upregulate or downregulate the BCR signal according to circumstances. They achieve this via the interaction of co-receptors with the BCR. Some of the main co-receptors are:
CD21: complement receptor type 2. Co-ligation of CD21 and the BCR by complement-coated antigen greatly enhances the BCR signal and allows B cells bearing low affinity receptors to be clonally expanded in the early stages of a developing immune response. This is the point at which the innate and adaptive immune systems interact.
CD22: a “siglec” (a lectin that binds to sialic acid residues). CD22 is constitutively associated with the BCR. It brings SHP-1 into the BCR complex. SHP-1 is a phosphatase that downregulates BCR-induced phosphorylation. If CD22 is excluded from the BCR, for example in the B cell synapse, BCR signalling is greatly upregulated. The biological role and physiological ligand(s) for CD22 are unknown.
FcgRIIb: This is the B cell’s receptor for IgG. If this receptor is co-ligated with the BCR, the BCR-induced Ca2+ signal and the ERK/MAPK pathways are turned off. Co-ligation of FcgRIIb with the BCR turns off immune responses and prevents over production of antibodies.
CD45: a transmembrane tyrosine phosphatase. The major role of CD45 is to positively regulate BCR signalling by dephosphorylating the inhibitory phosphate on src kinases.
PIR-B (LIR-1): “paired Ig-like receptor”. PIR-B has been described in mice. Its equivalent in humans is thought to be LIR-1 (leukocyte Ig-like receptor, also known as ILT-2). It has an ITIM motif and can recruit SHP-1 into the BCR. Its ligand and physiological role are unknown. It is possible that it is a receptor for MHC class I.

19. CD21 Antigen C3d CD21 CD225 (Leu-13) CD81 (Tapa-1) CD19
CD21 occurs in a complex with CD19, CD81 and CD225. CD21 binds to the C3d fragment of complement. Complement-coated antigens therefore can cross-link CD21 with the BCR.
Co-crosslinking of CD21 with the BCR introduces more CD19 into the BCR. This in turn allows more PI3-K and vav to be recruited, thus increasing the signal through the BCR.
CD81 (Tapa-1)
CD19

20. * * * * * CD22 SHP-1 CD79a/CD79b lyn
CD22 is constitutively associated with the BCR. CD22 has three ITIMs in its cytoplasmic domain
Following BCR ligation, CD22 is phosphorylated by lyn. The phosphorylated ITIMs recruit SHP-1. SHP-1 dephosphorylates lyn and the CD79a/CD79b ITAMs. CD22 therefore raises the threshold for signalling through the BCR.
CD22 is restricted to B cells. On resting cells it is “masked” i.e. it is bound to sialic acid residues on the surface of the cell it is expressed on. CD22 can be unmasked following cellular activation.
If CD22 is recruited away from the BCR, e.g. when B cell synapses form, then the threshold for signalling is lowered because negative regulators (SHP-1) are taken away from the BCR. * * *
SHP-1
CD79a/CD79b

21. * * * FcgRIIb Ras Antigen-Antibody Complex PIP3 PI(3,4)P2 SHIP Dok
lyn *
SHIP
Co-ligation of FcgRIIb and the BCR causes lyn to phosphorylate the ITM on FcgRIIb. The phosphorylated ITIM recruits SHIP. SHIP dephosphorylates PIP3, turning off PIP3-mediated signals such as Ca2+ flux.
SHIP also acts as an adaptor molecule. It recruits another adaptor, Dok, which in turn recruits and activates RasGAP. RasGAP inhibits the Ras pathway, turning off ERK and MAPK activity.
Dok
RasGAP
Ras

22. CD45
CD45
csk
lyn
CD45 is a transmembrane tyrosine phosphatase. It is constitutively associated with the BCR. Its function is to regulate the activity of lyn.
Csk (C-terminal src kinase) phosphorylates src kinases such as lyn on a negative regulatory residue in the C-terminus. Phosphorylation of this residue inactivates the src kinase. CD45 dephosphorylates the negative regulatory residue, thus allowing lyn to become active. Therefore the balance of activity between csk and CD45 regulates the signalling threshold through the BCR.

23. * * * PIR-B (LIR-1) btk syk SHP-1 SHP-2 lyn
PIR-B contains an ITIM that is phosphorylated by lyn. Phosphorylation of the ITIM causes recruitment and activation of the tyrosine phosphatases SHP-1 and SHP-2. These in turn inactivate syk and btk.

24. References
FASEB Journal 15: B cell receptor signalling and autoimmunity
Curr. Opin. Immunol. 13: New views of BCR structure and organisation
Trends in Immunology 22: Oligomeric antigen receptors: a new view on signalling for the selection of lymphocytes
Curr. Opin. Immunol. 12: Tec family kinases in lymphocyte signalling and function
Nature Immunology 1: Antibody regulation of B cell development
Immunological Reviews 176: Targets of B-cell antigen receptor signalling: the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase-3 signalling pathway and Rap1 GTPase
Immunological Reviews 176: Regulation of the phospholipase C-g2 pathway in B cells
Curr. Opin. Immunol. 13: Face off – the interplay between activating and inhibitory immune receptors
Science 290: Immune inhibitory receptors
Ann. Rev. Immunol. 18: Regulation of B lymphocyte responses to foreign and self-antigens by the CD19/CD21 complex
Nature Immunology 2: The immunological relay race: B cells take antigen by the synapse
Nature Immunology 2: CD45: new jobs for an old acquaintance
Semin. Immunol. 14: Cell surface immunoglobulin receptors in B cell development
Semin. Immunol. 14: Phosphoinositide 3-kinase in immunological systems