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B Cells and B Cell Development

Dr. Colin R.A. Hewitt


crah1@le.ac.uk

The discovery of B cell immunity


1954 - Bruce Glick, Ohio State University
Studies on the function of the bursa of Fabricius, a lymphoid organ in
the cloacal region of the chicken

Bursectomy no apparent effect


Bursectomised chickens
were later used in
experiments to raise
antibodies to Salmonella
antigens

None of the
bursectomised
chickens made
anti-Salmonella
antibodies

Bursa was later found to be the organ in which antibody producing cells
developed antibody producing cells were thereafter called B cells
Mammals do not have a bursa of Fabricius

Origin of B cells and organ of B cell


maturation
Transfer marked foetal
liver cells

Normal bone marrow

Mature marked
B cells
in periphery

No Mature
B cells
Defective bone marrow

B cell development starts in the foetal liver


After birth, development continues in the bone marrow

B cell development in the bone marrow

Regulates construction of an antigen receptor

Ensures each cell has only one specificity

Checks and disposes of self-reactive B cells

Exports useful cells to the periphery

Provides a site for antibody production

Bone Marrow provides a


MATURATION & DIFFERENTIATION MICROENVIRONMENT
for B cell development

Bone Marrow

S
M

M
E

Scheme of B Cell Development in the Bone Marrow


Progenitors

E
n
d
o
o
s
t
e
u
m
Stromal cells

Pre-B

X
X
X
Macrophage

Immature &
mature B
Central
Sinus

Bone marrow stromal cells nurture


developing B cells
1. Specific cell-cell contacts between stromal cells and developing B cells
2. Secretion of cytokines by stromal cells

Cell-cell contact

Secreted
Factors - CYTOKINES

Stromal cell

Types of cytokines and cell-cell contacts needed at


each stage of differentiation are different

Maturing B cells

Bone marrow stromal cell

B
B
Stromal cell

Stages of B cell development

Stem Cell

Early pro-B cell

Late pro-B cell

Large pre-B cell


Peripheral

Small pre-B cell

Immature B cell

Mature B cell

Each stage of development is defined by rearrangements of IgH


chain genes, IgL chain genes, expression of surface Ig,
expression of adhesion molecules and cytokine receptors

Cytokines and cell-cell contacts at each


stage of differentiation are different

VLA-4

Early
pro-B

Stem

(Integrin)

Receptor
Tyrosine
kinase

Stem cell
factor

VCAM-1
(Ig superfamily)

Cell adhesion
molecules

Kit

Cell-bound
growth
factor

Stromal cell

Cytokines and cell-cell contacts at each


stage of differentiation are different
Interleukin-7
receptor

Interleukin-7
Growth factor

Early
pro-B

Late
pro-B

Stromal cell

Pre-B

Stages of differentiation in the bone marrow are


defined by Ig gene rearrangement

B CELL STAGE

Stem cell

Early pro-B

IgH GENE
CONFIGURATION

Germline

DH to JH

Late pro-B Large pre-B


VH to DHJH

VHDHJH
Pre-B cell
receptor
expressed

Ig light chain gene has not yet rearranged

Pre- B cell receptor


Heavy chain
VHDHJH
Light chain
VLJLCL

VpreB
C H

5
Ig & Ig signal
transduction
molecules

Transiently expressed when VHDHJH CH is productively rearranged


VpreB/5 - the surrogate light chain, is required for surface expression
Ligand for the pre-B cell receptor may be galectin 1, heparan sulphate, other
pre-BCR or something as yet unknown

Ligation of the pre-B cell receptor


1. Suppresses further H chain rearrangement
2. Triggers entry into cell cycle
Large
Pre-B

Unconfirmed ligand
of pre-B cell receptor
1. Ensures only one specificty of
Ab expressed per cell

Stromal cell

2. Expands only the pre-B


cells with in frame VHDHJH joins

ALLELIC EXCLUSION
Expression of a gene on one chromosome prevents expression of the
allele on the second chromosome

Evidence for allelic exclusion


ALLOTYPE- polymorphism in the C region of Ig one
allotype inherited from each parent
Allotypes can be identified by staining B cell surface Ig with antibodies

(Refer back to Dreyer & Bennet hypothesis in Molecular Genetics


of Immunoglobulins lecture topic)

AND b

Suppression of H chain rearrangement by


pre-B cell receptor prevents expression of two
specificities of antibody per cell

a/b

b/b

a/a

Allelic exclusion prevents unwanted responses


One Ag receptor per cell

IF there were two Ag receptors per cell

Y
Y
YB

Y
YB

Self antigen
expressed by
e.g. brain cells

YY

Anti
brain
Abs

Y Y
Y Y
Y
Anti
S. aureus
Antibodies

Y
Y

S. aureus

Y Y
Y Y
Y

YY
S. aureus

Anti
S. aureus
Antibodies

Suppression of H chain gene rearrangement


ensures only one specificity of Ab expressed per cell.
Prevents induction of unwanted responses by pathogens

Allelic exclusion is needed for efficient clonal selection

Antibody
S. typhi

S. typhi

All daughter cells must express the same Ig specificity


otherwise the efficiency of the response would be compromised
Suppression of H chain gene rearrangement helps prevent the emergence of
new daughter specificities during proliferation after clonal selection

Allelic exclusion is needed to prevent holes in the repertoire


One specificity of Ag
receptor per cell

IF there were two specificities


of Ag receptor per cell

Anti-brain Ig

Exclusion of anti-brain B cells


i.e. self tolerance

B
Deletion

OR

Anergy

BUT anti S.aureus B cells


will be excluded
leaving a hole in the
repertoire

Anti-brain Ig
AND
anti-S. aureus Ig

S. aureus

Ligation of the pre-B cell receptor


1. Suppresses further H chain rearrangement
2. Triggers entry into cell cycle
Large
Pre-B

Unconfirmed ligand
of pre-B cell receptor
1. Ensures only one specificity of
Ab expressed per cell

Stromal cell

2. Expands only the pre-B


cells with in frame VHDHJH joins

Large pre-B cells need in frame VHDHJH joins to mature


Human IgG3 Heavy Chain
nucleotide sequence

Translation in frame 1

ATGAAACANCTGTGGTTCTTCCTTCTCCTGG
TGGCAGCTCCCAGATGGGTCCTGTCCCAGG
TGCACCTGCAGGAGTCGGGCCCAGGACTGG
GGAAGCCTCCAGAGCTCAAAACCCCACTTGG
TGACACAACTCACACATGCCCACGGTGCCCA
GAGCCCAAATCTTGTGACACACCTCCCCCGT
GCCCACGGTGCCCAGAGCCCAAATCTTGTG
ACACACCTCCCCCATGCCCACGGTGCCCAG
AGCCCAAATCTTGTGACACACCTCCCCCGTG
CCCNNNGTGCCCAGCACCTGAACTCTTGGG
AGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGATACCCTTATGATTTCCCGGACCC
CTGAGGTCACGTGCGTGGTGGTGGACGTGA
GCCACGAAGACCCNNNNGTCCAGTTCAAGT
GGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCTGCGGGAGGAGCAGTACA
ACAGCACGTTCCGTGTGGTCAGCGTCCTCA
CCGTCCTGCACCAGGACTGGCTGAACGGCA
AGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCC
AAAGCCAAAGGACAGCCCGAGGAGATGACC
AAGAACCAAGTCAGCCTGACCTGCCTGGTCA
AAGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAACACCACGCCTCCCATGCTGGACTCC
GACGGCTCCTTCTTCCTCTACAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACATCTTCTCATGCTCCGTGATGCATGAGGC
TCTGCACAACCGCTACACGCAGAAGAGCCTC
TCCCTGTCTCCGGGTAAATGA

MKXLWFFLLLVAAPRWVLSQV
HLQESGPGLGKPPELKTPLGD
TTHTCPRCPEPKSCDTPPPCP
RCPEPKSCDTPPPCPRCPEPK
SCDTPPPCXXCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDXXVQFKWYVDG
VEVHNAKTKLREEQYNSTFRV
VSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPEE
MTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYNTTPPMLD
SDGSFFLYSKLTVDKSRWQQG
NIFSCSVMHEALHNRYTQKSL
SLSPGK*

Translation in frame 2
(no protein)

Development
continues
Large
pre-B

Pre-B cell
receptor can
be activated

Development
arrests

*
Translation in frame 3
ETXVVLPSPGGSSQMGPVPGA
PAGVGPRTGEASRAQNPTW*

Development
arrests

Ligation of the pre-B cell receptor triggers


entry into the cell cycle

Proliferation

Large
Pre-B
Large
Large
Pre-B
Large
Pre-B
Large
Pre-B
Pre-B

Small
Large
pre-B

Proliferation
stops
Pre-receptor
not displayed

Many large pre-B


cells with identical
pre-B receptors

Large
pre-B

Large
Pre-B
Large
Large
Pre-B
Large
Pre-B
Large
Pre-B
Pre-B

IgM
Intracellular VDJCH chain
VL-JL rearranges

Immature
B cell

Light chain expressed


IgM displayed on surface

Heavy and light chain rearrangement is potentially wasteful


V

Germline

D D J

DH-JH joining

VH-DHJH joining

D J

Large
pre-B

With two random joins to generate a heavy chain


there is a 1:9 chance of a rearrangement of being in frame
V

Germline

V V

VL-JL joining

With one random join to generate a light chain


there is a 1:3 chance of a rearrangement being of frame

Small
pre-B

There is, therefore, only a 1:27 chance of an in frame rearrangement

Out of frame rearrangements arrest further B cell maturation

B cells have several chances to successfully


rearrange Ig genes
Early Pro B
DH-JH
On first
chromosome

NO
DH-JH
On second
chromosome

Late Pro B
YES

YES

VH-DJH
On first
chromosome

NO
VH-DJH
On second
chromosome

Pre B
YES

on first
YES
chromosome

Y
B

YES
on first
chromosome

IgM

on second
chromosome

NO

NO

NO
NO

IgM

YES

NO
YES

Immature B

on second
chromosome

NO

YES

Y
B

Acquisition of antigen specificity creates a


need
to check for recognition of self antigens

Small pre-B cell

Immature B cell

No antigen receptor at cell surface


Cell surface Ig expressed
Unable to sense Ag environment
Able to sense Ag environment
!!May be self-reactive!!
Can now be checked for self-reactivity
1. Physical removal from the repertoire
2. Paralysis of function
3. Alteration of specificity

DELETION
ANERGY
RECEPTOR EDITING

B cell self tolerance: clonal deletion

Small
pre-B

B
Immature
B

YY
Small pre-B cell
assembles Ig

Immature
B cell recognises
MULTIVALENT
self Ag

Clonal deletion by
apoptosis

B cell self tolerance: anergy


IgD normal IgM low

Immature
B

IgD

IgD

Small
pre-B

YY

IgM

IgD

B
Small pre-B cell
assembles Ig

Immature
B cell recognises
soluble self Ag
No cross-linking

Anergic B cell

Receptor editing
A rearrangement encoding a self specific receptor can be replaced
V

D J

!!Receptor
recognises
self antigen!!

Arrest development
And reactivate
RAG-1 and RAG-2

B
V

D J

Apoptosis
or anergy

Edited receptor now recognises


a different antigen and can be
rechecked for specificity

B cell self tolerance: export of self tolerant B cells

YY

Immature
B

IgD

IgM

Small pre-B cell


assembles Ig

Immature
B cell doesnt
recognise any
self Ag

YY
B

YY

YY

YY

Small
pre-B

IgD and IgM normal


IgD
IgM
IgM
IgD

IgM

IgD

Mature B cell
exported to the
periphery

How can B cells express


IgM and IgD simultaneously?
C

C3

C1

C1

C2

C4

C
C

C2
S3
C3

C3

S1
C1

VDJ

C3

VDJ

C1

VDJ

C1

VDJ

C3

VDJ

C1

VDJ

C1

IgG3 produced.
Switch from IgM

IgA1 produced.
Switch from IgG3

IgA1 produced.
Switch from IgM

N.B. Remember Molecular Genetics of Immunoglobulins lecture No C switch region


Consider similarities with mechanism allowing secreted and membrane Ig by the same cell

Splicing of IgM and IgD RNA


VD J

VD J

C1

C2

C3

C3

C1

C4

C1

C2

C3

pA2

pA1

DNA

Two types of mRNA can be made simultaneously in the cell by differential usage of
alternative polyadenylation sites and splicing of the RNA

VD J

C1

C2

C3

C4

AAA C1

V D J C

C2

C3

RNA cleaved and


polyadenylated at pA1

IgM mRNA
RNA cleaved and
polyadenylated at pA2

VD J

C1

C2

C3

C4

C1

C2

C3

pA1

V D J C

IgD mRNA

AAA

Summary
B cells develop in the foetal liver and adult bone marrow
Stages of B cell differentiation are defined by Ig gene
rearrangement
Pre-B cell receptor ligation is essential for B cell development
Allelic exclusion is essential to the clonal nature of immunity
B cells have several opportunities to rearrange their antigen
receptors
IgM and IgD can be expressed simultaneously due to differential
RNA splicing
So far, mostly about B cells in the bone marrow - what about mature
peripheral B cells?

What are the external signals that activate B cells?


Fab anti-mIg
mIg

(Fab)2 anti-mIg

Anti-(Fab)2

Although Fab fragments bind to


membrane Ig (mIg), no signal is
transduced through the B cell
membrane

Bridging (or Cross-linking) of


different mIg allows the B cell
receptor to transduce a weak signal
through the B cell membrane

Extensive cross linking of (Fab)2


bound to mIg using an anti-(Fab)2
antibody enhances the signal
through the B cell membrane

Ring staining

Patching

Ig is evenly distributed
around the cell surface

Ig is aggregated in
uneven clumps as
a result of mild
cross-linking of Ig

Capping
Ig is collected at a pole of the cell
in a cap as a result of extensive
cross-linking of Ig

Transduction of signals by the B cell receptor


Extracellular antigen
recognition domains

Ig Ig

The cytoplasmic domains of the Ig and


Ig contain Immunoreceptor Tyrosine
-based Activation Motifs (ITAMS) - 2
tyrosine residues separated by 9-12
amino acids - YXX[L/V]X6-9YXX[L/V]
Intracytoplasmic
signalling domains

Phosphorylation by Src kinases

Kinase domain

Enzyme domain
phosphorylates tyrosines
(to give phosphotyrosine)

SH2 domain SH3 domain Unique region

Phosphotyrosine
receptor domain

Adaptor
protein
recruitment
domain

ITAM
binding
domain

Phosphorylation changes the properties of a protein by changing its conformation


Changes in conformation may activate or inhibit a biochemical activity or create a
binding site for other proteins
Phosphorylation is rapid, requires no protein synthesis or degradation to change
the biochemical activity of a target protein
It is reversible via the action of phosphatases that remove phosphate

Regulation of Src kinases

Kinase domain

Inhibitory tyrosine residue

SH2 domain SH3 domain Unique region

Activating tyrosine residue

Phosphorylation of Activating Tyrosine stimulates kinase activity

Kinase domain

SH2 domain SH3 domain Unique region

Phosphorylation of Inhibitory Tyrosine inhibits kinase activity


by blocking access to the Activating Tyrosine Residue

Regulation of Src kinases by Csk and CD45


C terminus
Kinase domain

SH2 domain

SH3 domain Unique region

Activated cells: a
phosphatase associated
with the Leukocyte Common
Antigen - CD45, removes
the C terminus phosphate
allowing the activating
tyrosine to be
phosphorylated

Resting cells: Src


kinase is inactivated
by a constitutively
expressed C
-terminal Src kinase (Csk)

Kinase domain

Kinase domain

SH2 domain

SH3 domain

Unique region

The balance between Csk and CD45 phosphatase activity sets the threshold for
initiating receptor signalling

Phosphorylation of ITAMs by Src kinases

ITAM

ITAM

ITAM

P
ITAM

ITAM

P
1. Csk inactived
Src interacts
with low affinity
with the ITAMs
of resting
receptors

2. Antigen clusters B cell receptors with


CD45 phosphatases.
Src kinases are phosphorylated
and are activated to phosphorylate
ITAMS

3. Src kinases bind to


phosphorylated ITAMS
and are activated to
phosphorylate adjacent
ITAMS

Syk protein Tyrosine kinases


CD45 phosphatase allows activation of Src family kinases Blk, Fyn & Lyn
Receptor cross-linking activates Src kinases that phosphorylate ITAMs in
the Ig and Ig

ITAM

P
P

Syk - 2 x SH2
domains spaced to
bind to two
phosphotyrosines on
an ITAM

ITAM

ITAM

One Syk binds to Ig, one to Ig - each Syk


transphosphorylates the other

The B cell co-receptor


CD21
(C3d receptor)

CD45

CD19
Ig Ig
CD81
(TAPA-1)

The B cell co-receptor

Co-receptor phosphorylation
C3d opsonised bacterium

C3d binds to
CD21, the
complement
receptor 2
(CR2)

Antigen
recognition

P P

Src family kinases


then bind the
phosphorylated
CD19

mIg and CD21 are cross-linked by antigen that has activated complement
CD21 is phosphorylated and receptor-associated kinases phosphorylate CD19
Phosphorylated CD19 activates more Src family kinases
Ligation of the co-receptor increases B cell receptor signalling 1000 -10,000 fold

Activation of signals that affect gene transcription


BLNK

P P P P
Tec Tec Tec Tec

Cell membrane-associated B
cell Linker protein - BLNK contains many Tyrosine
residues
BLNK binds Tec kinases

ITAM

ITAM

Activated Syk phosphorylates BLNK


Activated Syk phosphorylates
Guanine-nucleotide exchange factors
(GEFS) that in turn activate small GTP
binding proteins Ras and Rac

Tec kinases activate


phospholipase C- (PLC-)

PLC- cleaves
phosphotidylinositol
bisphosphate (PIP2) to yield
diacylglycerol (DAG) and
inositol trisphosphate (IP3)
Ras and Rac activate the MAP
kinase cascade

Transmission of signals from the cell


surface to the nucleus
B cell-specific parts of the signalling cascade are associated with receptors
unique to B cells - mIg, CD19 etc.
Subsequent signals that transmit signals to the nucleus are common to
many different types of cell.
The ultimate goal is to activate the transcription of genes, the products of
which mediate host defence, proliferation, differentiation etc.

Once the B cell-specific parts of the cascade are complete, signalling to


the nucleus continues via three common signalling pathways via:
1.The mitogen-activated protein kinase (MAP kinase) pathway
2. Increased in intracellular Ca2+ mediated by IP3
3.The activation of Protein Kinase C mediated by DAG

Simplified scheme linking antigen recognition


with transcription of B cell-specific genes
MAP Kinase cascade
Small G-protein-activated MAP kinases found in all multicellular animals activation of MAP kinases ultimately leads to phosphorylation of transcription
factors from the AP-1 family such as Fos and Jun.
Increases in intracellular calcium via IP3
IP3, produced by PLC-, binds to calcium channels in the ER and releases
intracellular stores of Ca++ into the cytosol. Increased intracellular [Ca++]
activate a phospatase, calcineurin, which in turn activates the transcription
factor NFAT.
Activation of Protein Kinase C family members via DAG
DAG stays associated with the membrane and recruits protein kinase C
family members. The PKC, serine/threonine protein kinases, ultimately
activate the transcription factor NFB

The activated transcription factors AP-1, NFAT and NFB induce B cell
proliferation, differentiation and effector mechanisms

YY

YY
Mature peripheral
B cell

YY

YY

B cell recognises
non-self antigen
in periphery

Y
Y
Y

YY
YY
YY

YY

YY

Differentiation in the periphery

Ig-secreting plasma cell

Plasma cells
Surface Surface High rate Growth Somatic Isotype
Ig
MHC II Ig secretion
hypermutn switch

High

Yes

No

Yes

Yes

Low
No

No

Yes

No

No

Mature B cell

B
Plasma cell

Yes

Summary
You should know:
Where B cells come from
What happens to B cells in the bone marrow
How B cell differentiation is linked with Ig gene rearrangement
The B cell developmental check points that ensure each cell
produces a single specificity of antibody that does not react with
self
How B cells transmit information from the shape and charge of an
antigen through the cell membrane to allow the expression of
genes in the nucleus

What do mature B cells do once activated by an


antigen in the periphery?

Recirculating B cells normally pass through


lymphoid organs
T cell area

B cells in
blood

B cell
area

Efferent
lymph

Recirculating B cells are trapped by foreign


antigens in lymphoid organs

Antigen enters
node in afferent
lymphatic

YY
Y

B cells
proliferate
rapidly

B cells leave blood &


enter lymph node via
high endothelial venules

Y
Y
YY
YY

Y
YYY
Y
Y

GERMINAL CENTRE
Transient structure of
Intense proliferation

YY
Y

Germinal centre
releases B cells
that differentiate
into plasma cells

B cells (stained brown) in the Germinal Centre

P = Paracortex, Mn = Mantle zone SC = Subcapsular zone

Follicular Dendritic cells (stained blue)in the Germinal Centre

Retention of Antigens on Follicular Dendritic Cells


Radiolabelled antigen localises on the surface of Follicular Dendritic cells
and persists there, without internalisation, for very long periods

Maturation of Follicular Dendritic cells

Club-shaped tips of developing dendrites

Filiform dendrites

Bead formation on dendrites

Bead formation on dendrites

Association of antigen with FDC


Antigen enters the germinal centre
in the form of an immune complex with
C3b and antibodies attached

The Immune complexes bind to


Fc and complement receptors
on the FDC dendrites

Ig Fc receptor

Complement receptor 3

FDC surface
The filiform dendrites of FDC develop
beads coated with a thin layer of immune
complexes

Iccosome formation and release

DC veils

The veils of antigen-bearing dendritic cell


surround the beads and the layer of
immune complexes is thickened by
transfer from the dendritic cell.
These beads are then released and are
then called ICCOSOMES

Iccosomes (black coated particles)


bind to and are taken up by B cell
surface immunoglobulin

Uptake of Iccosomes/Antigen by B cells

Anti-

Y
Y
Y

Iccosomes bearing
different antigens

Surface Ig captures antigen


Cross-linking of antigen receptor activates B cell
Activated B cell expresses CD40

B cell

B
CD40

Fate of Antigens Internalised by B cells

1. Capture by antigen
specific Ig maximises
uptake of a single antigen

2. Binding and internalisation via


Ig induces expression
of CD40
3. Antigen enters exogenous antigen
processing pathway
4. Peptide fragments of antigen are loaded
onto MHC molecules intracellularly.
MHC/peptide complexes are
expressed at the cell surface

T cell help to B cells


Signal 2 - T cell help

YYY

Signal 1
antigen & antigen
receptor

Th
Th
1. T cell antigen receptor
2. Co-receptor (CD4)
3.CD40 Ligand

T cell help - Signal 2

Signal 2

Cytokines

Th

IL-4
IL-5
IL-6
IFN-
TGF-

Cytokines

YYY
Signal 1

B cells are inherently prone to die by apoptosis


Signal 1 & 2 upregulate Bcl-XL in the B cell and
Bcl-XL prevents apoptosis
Signal 1 & 2 thus allow the B cell to survive
T cells regulate the survival of B cells and thus
control the clonal selection of B cells

T cell help - Signal 2 activates hypermutation


Signal 2

Receipt of signal 2
by the B cell also
activates
hypermutation in the
CDR - encoding
parts of the Ig genes

Th

Signal 1

Day 6

Day 8

Day 12

Day 18

Clone 1
Clone 2
Clone 3
Clone 4
Clone 5
Clone 6
Clone 7
Clone 8
Clone 9
Clone 10

Signal 2, and thus T cells,


regulate which B cells are
clonally selected.
Low affinity Ig takes up and
presents Ag to T cells
inefficiently.
Inefficient presentation to T
cells does not induce CD40.

Deleterious mutation
Beneficial mutation
Neutral mutation

CDR3

CDR1
CDR2

CDR3

CDR1
CDR2

CDR3

CDR1
CDR2

CDR3

CDR1
CDR2

With no signal 2 delivered


by CD40, low affinity B cells
die.

Lower affinity - Not clonally selected


Higher affinity - Clonally selected
Identical affinity - No influence on clonal selection

Only B cells with high affinity


Ig survive - This is affinity
maturation

Control of Affinity & Affinity Maturation


Five B cell antigen
receptors all specific
for
, but with
different affinities
due to somatic
hypermutation
of Ig genes in
the germinal centre

Only this cell, that has a high affinity for antigen can express CD40.
Only this cell can receive signal 2
Only this cell is rescued from apoptosis i.e. clonally selected
The cells with lower affinity receptors die of apoptosis by neglect

Germinal Centre Macrophages (stained brown)


Clean Up Apoptotic Cells

GC = Germinal Centre, TBM = Tingible Body Macrophages

Role of T cell cytokines in T cell help


Signal 2

Cytokines
IL-4
IL-5
IL-6
IFN-
TGF-

Th

YYY
Signal 1

B
B B
B
B

B
B
B
B
B

PC

B
B
B

Proliferation & Differentiation


IgM

IgG3

IgG1

IgG2b IgG2a IgE

IgA

IL4
inhibits inhibits induces
inhibits induces
IL-5
augments
IFN- inhibits induces inhibits
induces inhibits
TGF- inhibits inhibits
induces
induces

Regulation of specificity - Cognate recognition


1. T cells can only help the B cells that present
antigen to them
2. B cells are best at presenting antigens that they
take up most efficiently
3. B cells are most efficient at taking up antigens that
their B cell antigen receptors bind to
4. T and B cells help each other to amplify immunity
specific for the same antigen

i.e. Regulates the Characteristics of Adaptive Immunity


Sharply focuses specificity - Pathogen specificity
Improves specificity & affinity - Better on 2nd exposure
Is specific antigen dependent - Learnt by experience
Seeds memory in T and B cell pools

Synaptic tethering of a B cell (red)


to a T cell (green)

T cell (in centre) surrounded by B cells with


cytoskeleton stained green

Recirculating B cells are trapped by foreign


antigens in lymphoid organs

Antigen enters
node in afferent
lymphatic

YY
Y

B cells
Rapidly
proliferate
in follicles

B cells leave blood &


enter lymph node via
high endothelial venules

Y
Y
YY
YY

Y
YYY
Y
Y

GERMINAL CENTRE
Transient structure of
Intense proliferation

YY
Y

Germinal centre
releases B cells
that differentiate
into plasma cells

B cells (90%) and T cells


(10%) migrate to form
a primary follicle
B

B
T
B
B
B

1. Antigen loaded dendritic cells


migrate from subcapsular
sinus to paracortical area
of the lymph node

Primary follicle
formation
3. T cells
proliferate
B B
B T T
B T T T
B B

4. B cells migrate through


HEV - most pass through the B
paracortex and primary follicle.
Some interact with T cells and
proliferate to form a primary focus

DC
T
T

HEV

2. T cells migrate
through HEV and
are trapped by
antigen on DC

T cell motility in the lymph node

Germinal Centre Microanatomy


2. B cells (centrocytes) upregulate
surface Ig, stop dividing and
receive costimulatory signals
from T cells and FDC

Light zone

4. Selected cells leave lymph


node as memory
cells or plasma cells

Primary Follicles become


secondary
follicles
B
Dark zone
when germinal centres develop
T

Follicular dendritic
cells select useful
B cells
3. Apoptosis of
self-reactive &
unselected cells

1. B cells (centroblasts) downregulate


surface Ig, proliferate, somatically
hypermutate their Ig genes.
AFFINITY MATURATION

Two B cell lineages


B cell precursor

Mature B cell

Plasma cell

PC
B2 B cells

Y
Y
Y

YY
YYYY

?
CD5

Y
Y
Y
Y

Y
Y

Y
Y

Distinct B cell
precursor

IgG

B1 B cells
Primitive B cells found in
pleura and peritoneum

IgM - no other isotypes

B-1 B Cells
IgM uses a distinctive & restricted range of V regions

CD5

Y
Y
Y
Y

Y
Y

Y
Y

Few non-template encoded (N) regions in the IgM

IgM

Recognises repeating epitope Ag such as


phospholipid phosphotidyl choline &
polysaccharides
NATURAL ANTIBODY
NOT part of adaptive immune response:
No memory induced
Not more efficient on 2nd challenge
Present from birth
Can make Ig without T cell help

Comparison of B-1 and B-2 B cell properties


Property
N regions
V region repertoire
Location
Renewal
Spontaneous Ig production
Isotypes
Carbohydrate
specificity
Carbohydrate specificity
Protein specificity
Protein
specificity
Need T cell
cellhelp
help
Somatic hypermutation of Ig
Memory development

B-1 cells
Few
Restricted
Peritoneum/pleura
Self renewal in situ
High
IgM
Yes
Yes
Rarely
Rarely
No
No
No
No

B-2 cells
Extensive
Diverse
Everywhere
Bone marrow
Low
IgM/G/A/D/E
Rarely
Yes
Yes
Yes
Yes
High
Yes

Specificity & requirement for T cell help suggests strikingly different types
of antigens are seen by B-1 and B-2 B cells

T Independent Antigens (TI-2)

Y
Mature
B-1

Y
Y
Y
Y

Y
Y

Y
Y

YY
B-2 cell repertoire is purged
of cells recognising
multivalent antigens during
development in the bone
marrow

Y
Y
Y

Immature
B-2 Cell

Immature B cells that bind to


multivalent self Ag undergo
apoptosis

TI-2 Antigen

IgM
Non-bone marrow derived B-1 cells
are directly stimulated by antigens
containing multivalent epitopes.
No T cells are necessary
Induces the expression of natural
antibodies specific for TI-2 antigens

T Independent Antigens (TI-1)


LPS binding
protein
LPS
TLR 4

Bacterial Lipopolysaccharides, (TI-1


antigens), bind to host LPS binding
protein in plasma
LPS/LPSBP is captured by CD14
on the B cell surface
Toll - like receptor 4 (TLR4) interacts
with the CD14/LPS/LPSBP complex

CD14

B Cell

Activation of B cell

T Independent Antigens (TI-1 e.g. LPS)


LPS complexes with CD14, LPSBP & TLR4

Y Y Y Y Y Y

Six different B cells will require 6 different antigens to activate them


At high dose TI-1 antigens (like LPS) will POLYCLONALLY ACTIVATE all of
the B cells irrespective fo their specificity.
TI-1 antigens are called MITOGENS

Y YY YY YY YY YY Y
YY YY YY YY YY YY
YY YY YY YY YY YY

T Dependent & Independent Antigens

Induce responses in babies


Induce responses in athymics
Prime T cells
Polyclonally activate B cells
Require repeating epitopes

T
Dependent
Antigens

TI-1
Antigens

TI-2
Antigens

Yes
No
Yes
No
No

Yes
Yes
No
Yes
No

No
Yes
No
No
Yes

Why are babies unresponsive to TI-2 antigens?


In adults:

TI-2 Antigen

Y
Mature
B-1

Y
Y
Y
Y

Y
Y

Y
Y

YY
Adult immature B cells that
bind to multivalent self Ag
undergo apoptosis

Y
Y
Y

Immature
B-2 Cell

IgM
Adult non-bone marrow derived B-1
cells are directly stimulated by
antigens containing multivalent
epitopes produce IgM WITHOUT T
cell help.

Why are babies unresponsive to TI-2 antigens?


In babies:
All B cells, B-1 & B-2, are immature
TI-2 Antigen
Immature
B-1 Cell

Immature B cells that bind to


multivalent self Ag undergo
apoptosis

YY

As with adult B cells, immature B cells that bind multivalent self Ag


undergo apoptosis
Hence babies do not respond to TI-2 antigens.
Babies are, therefore susceptible to pathogens with multivalent
antigens such as those on pneumococcus

T Dependent & Independent Antigens

Induces response in babies


Induces response in athymia
Primes T cells
Polyclonally activates B cells
Requires repeating epitopes

T
Dependent
Antigens

TI-1
Antigens

TI-2
Antigens

Yes
No
Yes
No
No

Yes
Yes
No
Yes
No

No
Yes
No
No
Yes

TD: Activate B-1 and B-2 B cells


TI-1: Activate B-1 and B-2 B cells
TI-2: Activate only B-1 B cells

Examples
TD: Diptheria toxin, influenza heamagglutinin, Mycobacterium tuberculosis
TI-1: Bacterial lipopolysaccharides, Brucella abortis
TI-2: Pneumococcal polysaccharides, Salmonella polymerised flagellin

Immune effector mechanisms against


extracellular pathogens & toxins
NEUTRALISATION
`

Bacterium

Toxin

Toxin release
blocked

Prevents
invasion

Prevents
toxicity

Adhesion to
host cells blocked

NEUTRALISING ANTIBODIES

Effector mechanisms against


extracellular pathogens
OPSONISATION
Bacteria in extracellular space

+
Ab

OPSONISATION

Fc receptor
binding

Phagocytosis

Effector mechanisms against


extracellular pathogens
COMPLEMENT Activation
Bacteria in plasma

Lysis

+
Ab &
COMPLEMENT

Opsonisation

Complement &
Fc receptor
binding

Phagocytosis

Summary

B cell tolerance of self is by clonal deletion or anergy of self-reactive cells


Receptor editing increases the efficiency of B cell development
Follicular dendritic cells acquire antigen and transfer it to B cells
T cell help to B cells is via CD40L and cytokines
CD40 expression indirectly leads to Ig affinity maturation
Germinal centre microanatomy & function
There are two lineages of B cells - B1 and B2 B cells
The dependency of B cells upon T cells varies

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