Immune receptor

signaling
 B Cell Receptor Signalling

Immunoglobulin molecule

CD79 (Ig) CD79 (Ig)

The surface Ig molecule plus one molecule each of CD79 and CD79
together form the B cell receptor for antigen (BCR). The Ig molecule has a
very short cytoplasmic tail and cannot signal by itself. CD79 and
CD79contain ITAMs and are the signal transducing parts of the receptor.
 Structure of the BCR

CD79 and CD79 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.
 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.
 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

* ** ** ** ** ** *
 Conventions in Signalling Diagrams

Symbol Meaning

A B A inhibits B

A B A activates B, or A is
converted to B

A B A binds to B
 BCR Signalling – the First Steps

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 CD79
and CD79.
The tyrosine kinase, syk, is associated with CD79 and CD79. 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 Lyn
to rafts where it phosphorylates Syk is recruited to
Lyn found Lyn
encounters lyn ITAMs phosphorylated
in rafts phosphorylates
ITAMs syk
 syk
CD79 CD79

PIP3
lyn * *
* *

PI3-K btk B PLC2

L
N
K
vav
PIP5K
rac

Lyn is responsible for initiating BCR signalling. Lyn phosphorylates PI3-kinase causing the production of PIP 3. 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 PLC2 (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.
 CD19
syk
CD79 CD79 CD79 CD79

* * PIP3
* *
* * * * PI3-K
*
**
vav
*
*
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 PIP 3 production.
 PtdIns 3 kinase and Ca2+
PtdIns(3,4,5)P3 targets both PLC and btk to the plasma membrane. Btk is
then phosphorylated by lyn, autophosphorylated by itself, and finally
phosphorylates and activates PLC. After 10 to 20 seconds, syk takes
over the role of lyn. The continued presence of PtdIns(3,4,5)P 3 and the
continued activity of syk are required to maintain a Ca 2+ signal. syk
Ca2+

Ins(1,4,5)P3
PLC btk lyn
PKC
DAG 

PtdIns(4,5)P2 PtdIns(3,4,5)P3

PtdIns 3 kinase
 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

Caspase 9 PKB PDK-1

(Akt)
Bad S6 kinase
GSK 3
 Vav: effects on the cytoskeleton

Ca2+ Dissociates profilin and gelsolin

from actin. Promotes interaction
between actin and the cytoskeleton
Ins(1,4,5)P3
proteins vinculin, talin and zyxin.
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
PtdIns 4 kinase PtdIns(4)P 5 kinase plasma membrane).
PtdIns 3 kinase

cdc42 rac1 RhoA

Vav
 Vav: effects on MAPkinases

vav

cdc42 rac1 RhoA

MEKK1 NB, this is not the major

pathway of vav action.
Stimulated by
PKC and Ca2+. MKK4 (Sek1) p38
The point of
action of these
molecule on this
kinase cascade Jnk1 (SAPK) MAPKAP kinase 2
is unknown.

homodimerisation C-jun; atf-2; p53; ets

Enters nucleus
 Ras Pathway

Ras GAP ras sos

PKC raf1

MEK1 MEK2

ERK1 ERK2
homodimerisation homodimerisation

nucleus Fos; jun; ets

 Shc regulates the ras pathway

PIP3 PI3-K

sos
Gab1
ras
shc ?
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 PIP 3 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.
 BCR Signalling in B Cell Development
anergy

Receptor editing
apoptosis
Pre-B cell receptor
Pro-B cell receptor
(Ig, 5, Vpre-B,
(calnexin, CD79, autoreactive
CD79, CD79)
CD79)

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.
Transitional B

Pro-BCR signalling may be required for transition to large pre-B stage.

This is ligand-independent.
Marginal zone B
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
B1 Mature B
a mechanism for eliminating autoreactive B cells.
BCR signalling on mature B cells leads to proliferation.
 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.
 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: 489-494 2001 and Nature Immunology 2: 480-482 2001.

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 Fc
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.

 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.

FcRIIb: 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 FcRIIb 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.
 Antigen

C3d CD21

CD21 CD225 CD21 occurs in a complex with CD19, CD81

(Leu-13) and CD225. CD21 binds to the C3d fragment
CD81 of complement. Complement-coated antigens
therefore can cross-link CD21 with the BCR.
CD19 (Tapa-1)

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.
 CD22

** lyn CD22 is constitutively associated with the BCR. CD22 has three ITIMs in its cytoplasmic

** domain

* SHP-1 Following BCR ligation, CD22 is phosphorylated by lyn. The phosphorylated ITIMs
recruit SHP-1. SHP-1 dephosphorylates lyn and the CD79/CD79 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
CD79/CD79 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.
Antigen-Antibody
Complex FcRIIb

** PIP3 PI(3,4)P2
lyn
* SHIP Co-ligation of FcRIIb and the BCR causes lyn to
phosphorylate the ITM on FcRIIb. The phosphorylated
ITIM recruits SHIP. SHIP dephosphorylates PIP 3, turning
Dok off PIP3-mediated signals such as Ca2+ flux.
Ras RasGAP
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.
 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.
 PIR-B (LIR-1)

syk
lyn ** SHP-1

* btk
SHP-2

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.
Antigen Binding → BCR clustering → Lyn/Syk activation →
↓
PLC-γ → Ca²⁺ → NFAT
↓
PI3K → Akt → Survival/Proliferation
↓
MAPK → AP-1 → Proliferation
↓
NF-κB → Gene transcription
↓
Outcome: Activation, proliferation, differentiation

Memorize this!
 References
FASEB Journal 15: 2085-98 2001 B cell receptor signalling and autoimmunity
Curr. Opin. Immunol. 13: 270-77 2001 New views of BCR structure and organisation
Trends in Immunology 22: 356-60 2001 Oligomeric antigen receptors: a new view on signalling for the selection of
lymphocytes
Curr. Opin. Immunol. 12: 282-88 2000 Tec family kinases in lymphocyte signalling and function
Nature Immunology 1: 379-85 2000 Antibody regulation of B cell development
Immunological Reviews 176: 47-68 2000 Targets of B-cell antigen receptor signalling: the phosphatidylinositol 3-
kinase/Akt/glycogen synthase kinase-3 signalling pathway and Rap1 GTPase
Immunological Reviews 176: 19-29 2000 Regulation of the phospholipase C- 2 pathway in B cells
Curr. Opin. Immunol. 13: 326-31 2001 Face off – the interplay between activating and inhibitory immune receptors
Science 290: 84-89 2000 Immune inhibitory receptors
Ann. Rev. Immunol. 18: 393-422 2000 Regulation of B lymphocyte responses to foreign and self-antigens by the
CD19/CD21 complex
Nature Immunology 2: 480-482 2001 The immunological relay race: B cells take antigen by the synapse
Nature Immunology 2: 389-396 2001 CD45: new jobs for an old acquaintance
Semin. Immunol. 14: 351-358 2002 Cell surface immunoglobulin receptors in B cell development
Semin. Immunol. 14: 7-18 2002 Phosphoinositide 3-kinase in immunological systems