B Cells and B Cell Development

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

None of the
bursectomised
Bursectomised chickens chickens made
were later used in anti-Salmonella
experiments to raise antibodies
antibodies to Salmonella
antigens

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

Mature marked
Transfer marked foetal B cells
liver cells in periphery
Normal bone marrow

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

B Regulates construction of an antigen receptor

B Ensures each cell has only one specificity

B Checks and disposes of self-reactive B cells

B Exports useful cells to the periphery

B Provides a site for antibody production

Bone Marrow provides a

MATURATION & DIFFERENTIATION MICROENVIRONMENT
for B cell development
Bone Marrow

S
M

E
Scheme of B Cell Development in the Bone Marrow
Immature &
Progenitors Pre-B mature B

X
Central
E Sinus
n
d
o
o
s
t
X
e
u
m
Stromal cells
X
Macrophage
 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

Secreted
Cell-cell contact Factors - CYTOKINES
B

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

Kit
Early Receptor
VLA-4 Stem pro-B Tyrosine
(Integrin) kinase

Stem cell
VCAM-1
(Ig superfamily)
factor
Cell-bound
growth
Cell adhesion factor
molecules
Stromal cell
Cytokines and cell-cell contacts at each
stage of differentiation are different

Interleukin-7
receptor
Interleukin-7
Growth factor

Late
Early pro-B Pre-B
pro-B

Stromal cell
 Stages of differentiation in the bone marrow are
defined by Ig gene rearrangement

B CELL STAGE Stem cell Early pro-B Late pro-B Large pre-B

IgH GENE
Germline DH to JH VH to DHJH VHDHJH
CONFIGURATION

Pre-B cell
receptor
expressed

Ig light chain gene has not yet rearranged

 Pre- B cell receptor
Heavy chain
VHDHJH

Light chain VpreB

VLJLCL
C Hµ λ5
Igα & Igβ signal
transduction
molecules

Transiently expressed when VHDHJH CHµ is productively rearranged

VpreB/λ5 - 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 Unconfirmed ligand

Pre-B of pre-B cell receptor

1. Ensures only one specificty of

Ab expressed per cell
2. Expands only the pre-B
cells with in frame VHDHJH joins
Stromal cell

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

a/a b/b a/b

Y
Y

Y
a B b B a B AND b B

Y Y
Suppression of H chain rearrangement by
pre-B cell receptor prevents expression of two
b B a
specificities of antibody per cell
(Refer back to Dreyer & Bennet hypothesis in Molecular Genetics
of Immunoglobulins lecture topic)
Allelic exclusion prevents unwanted responses
One Ag receptor per cell IF there were two Ag receptors per cell

Y
YBY Y
Self antigen YB
YY expressed by YY
e.g. brain cells

S. aureus
Y S. aureusY
Y
Y
Y Y

Y
Y Y Y Y
Anti
S. aureus YAnti
brain
Y Anti
S. aureus

Y Y Antibodies
Antibodies
Y Y Abs
Y Y
Y
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 IF there were two specificities

receptor per cell of Ag receptor per cell
Anti-brain Ig Anti-brain Ig

Y Y
AND
anti-S. aureus Ig
Y

Y
Y
B B
Exclusion of anti-brain B cells
i.e. self tolerance BUT anti S.aureus B cells
will be excluded
B B leaving a “hole in the
OR
repertoire”

Deletion Anergy
Y Y
Y
S. aureus
B
 Ligation of the pre-B cell receptor

1. Suppresses further H chain rearrangement

2. Triggers entry into cell cycle

Large Unconfirmed ligand

Pre-B of pre-B cell receptor

1. Ensures only one specificity of

Ab expressed per cell
2. Expands only the pre-B
cells with in frame VHDHJH joins
Stromal cell
 Large pre-B cells need in frame VHDHJH joins to mature
Human IgG3 Heavy Chain
nucleotide sequence
ATGAAACANCTGTGGTTCTTCCTTCTCCTGGT
GGCAGCTCCCAGATGGGTCCTGTCCCAGGT
GCACCTGCAGGAGTCGGGCCCAGGACTGGG
GAAGCCTCCAGAGCTCAAAACCCCACTTGGT
GACACAACTCACACATGCCCACGGTGCCCAG
AGCCCAAATCTTGTGACACACCTCCCCCGTG
CCCACGGTGCCCAGAGCCCAAATCTTGTGAC
ACACCTCCCCCATGCCCACGGTGCCCAGAG
CCCAAATCTTGTGACACACCTCCCCCGTGCC
CNNNGTGCCCAGCACCTGAACTCTTGGGAG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGATACCCTTATGATTTCCCGGACCCCT
GAGGTCACGTGCGTGGTGGTGGACGTGAGC
CACGAAGACCCNNNNGTCCAGTTCAAGTGGT
ACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCTGCGGGAGGAGCAGTACAACA
GCACGTTCCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAACGGCAAGGA
GTACAAGTGCAAGGTCTCCAACAAAGCCCTC
CCAGCCCCCATCGAGAAAACCATCTCCAAAG
CCAAAGGACAGCCCGAGGAGATGACCAAGA
ACCAAGTCAGCCTGACCTGCCTGGTCAAAGG
CTTCTACCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTA
CAACACCACGCCTCCCATGCTGGACTCCGAC
GGCTCCTTCTTCCTCTACAGCAAGCTCACCG
TGGACAAGAGCAGGTGGCAGCAGGGGAACA
TCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCGCTACACGCAGAAGAGCCTCTCC
CTGTCTCCGGGTAAATGA
Translation in frame 1 Development
continues
MKXLWFFLLLVAAPRWVLSQV
HLQESGPGLGKPPELKTPLGD
Large Pre-B cell
TTHTCPRCPEPKSCDTPPPCP
RCPEPKSCDTPPPCPRCPEPK
pre-B receptor can
SCDTPPPCXXCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTC be activated
VVVDVSHEDXXVQFKWYVDG
VEVHNAKTKLREEQYNSTFRV
VSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPEE
MTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYNTTPPMLD
SDGSFFLYSKLTVDKSRWQQG
NIFSCSVMHEALHNRYTQKSL
SLSPGK*
Development
Translation in frame 2
(no protein) arrests
*
Translation in frame 3 Development
ETXVVLPSPGGSSQMGPVPGA
PAGVGPRTGEASRAQNPTW*
arrests
 Ligation of the pre-B cell receptor triggers
entry into the cell cycle

Large Large
Pre-B
Large
Pre-B
Large Many large pre-B
Large Proliferation
Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B cells with identical
Large Large
pre-B Pre-B
Pre-B
Pre-B
Pre-B
pre-B receptors

Y
Small
Large Immature
pre-B
IgM
B cell

Proliferation
Intracellular VDJCH chain Light chain expressed
stops
Pre-receptor VL-JL rearranges IgM displayed on surface
not displayed
Heavy and light chain rearrangement is potentially wasteful

V D J C Germline

V D D J C DH-JH joining Large

pre-B
V V D J C VH-DHJH joining

With two “random” joins to generate a heavy chain

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

V J C Germline

V V J C VL-JL joining

With one “random” join to generate a light chain Small

there is a 1:3 chance of a rearrangement being of frame 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 Late Pro B Pre B Immature B
DH-JH YES VH-DJH κ on first
YES YES IgMκ
On first On first chromosome
chromosome
NO
YES
chromosome
NO YES
NO
κ on second
chromosome
YES
Y
B
DH-JH VH-DJH NO
On second On second YES
λ on first
chromosome chromosome
chromosome IgMλ
NO

NO
NO
λ on second
chromosome
YES
Y
B
B
NO
 Acquisition of antigen specificity creates a
need
to check for recognition of self antigens
Y

Y
Y
B BY
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

• Physical removal from the repertoire DELETION

• Paralysis of function ANERGY
• Alteration of specificity RECEPTOR EDITING
 B cell self tolerance: clonal deletion

Small
B
B
pre-B Immature

YY BB

Small pre-B cell Immature Clonal deletion by

assembles Ig B cell recognises apoptosis
MULTIVALENT
self Ag
 B cell self tolerance: anergy

IgD normal IgM low

Small
YY Y IgM

Y
Y
pre-B Immature IgD IgD
BB YB

IgD

B
Small pre-B cell Immature
assembles Ig 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 V V V D J C

!!Receptor

Y
B recognises
self antigen!!
Arrest development
And reactivate B Apoptosis
or anergy
RAG-1 and RAG-2

V V V D J C

Edited receptor now recognises

Y

B a different antigen and can be

rechecked for specificity
B cell self tolerance: export of self tolerant B cells

IgD and IgM normal

Small
YY IgM
IgD

YY IgM
B Immature

YY
pre-B
BB IgD

YY
IgD
YYB
IgM IgM
IgD

Mature B cell
Small pre-B cell Immature exported to the
assembles Ig B cell doesn’t periphery
recognise any
self Ag
 How can B cells express
IgM and IgD simultaneously?

Cµ Cδ Cγ3 Cγ1 Cα1 Cγ2 Cγ4 Cε Cα2

Sγ3
Cδ Cδ

Cγ3
Cµ Cγ3 Cµ Sγ1
Cγ1

VDJ Cγ3 VDJ Cα1 VDJ Cα1

VDJ Cγ3 VDJ Cα1 VDJ Cα1

IgG3 produced. IgA1 produced. IgA1 produced.

Switch from IgM Switch from IgG3 Switch from IgM
N.B. Remember Molecular Genetics of Immunoglobulins lecture – No Cδ switch region
Consider similarities with mechanism allowing secreted and membrane Ig by the same cell
 Splicing of IgM and IgD RNA

VDJ Cµ Cδ Cγ3 Cγ1

VDJ Cµ1 Cµ2 Cµ3 Cµ4 Cδ1 Cδ2 Cδ3 DNA

pA1 pA2
Two types of mRNA can be made simultaneously in the cell by differential usage of
alternative polyadenylation sites and splicing of the RNA

AAA Cδ1 RNA cleaved and

VD J Cµ1 Cµ2 Cµ3 Cµ4 Cδ2 Cδ3
polyadenylated at
pA1

V D J Cµ IgM mRNA
RNA cleaved and
polyadenylated at pA2
VDJ Cµ1 Cµ2 Cµ3 Cµ4 Cδ1 Cδ2 Cδ3 AAA
pA1

V D J Cδ IgD mRNA
 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 Although Fab fragments bind to

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

(Fab)2 anti-mIg 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

Anti-(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 Ig is aggregated in
around the cell surface 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

Igβ Igα
The cytoplasmic domains of the Igα and
Igβ 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 SH2 domain SH3 domain Unique region

Enzyme domain Phosphotyrosine Adaptor ITAM

phosphorylates tyrosines receptor domain protein binding
(to give phosphotyrosine) recruitment domain
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 SH2 domain SH3 domain Unique region

Inhibitory tyrosine residue 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 SH3 domain Unique region
SH2 domain

Resting cells: Src Activated cells: a

kinase is inactivated phosphatase associated
by a constitutively with the Leukocyte
expressed C Common Antigen - CD45,
-terminal Src kinase - removes the C terminus
(Csk) phosphate allowing the
activating tyrosine to be
phosphorylated
Kinase domain

Kinase domain SH3 domain Unique region

SH2 domain

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 P ITAM P
P
P

1. Csk inactived
Src interacts 3. Src kinases bind to
with low affinity 2. Antigen clusters B cell receptors with phosphorylated ITAMS
with the ITAMs CD45 phosphatases. and are activated to
of ‘resting’ Src kinases are phosphorylated phosphorylate adjacent
receptors and are activated to phosphorylate ITAMS
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 Igα and Igβ

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

One Syk binds to Igα, one to Igβ - each Syk

transphosphorylates the other
 The B cell co-receptor

CD21
(C3d receptor)
CD45

CD19

Igβ Igα
CD81
(TAPA-1)

The B cell co-receptor

 Co-receptor phosphorylation
C3d opsonised bacterium

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

Src family kinases

P P P P 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

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

P P Tec kinases activate

ITAM ITAM
phospholipase C- γ (PLC-γ)
P P
PLC-γ cleaves
P P phosphotidylinositol
bisphosphate (PIP2) to yield
diacylglycerol (DAG) and
Activated Syk phosphorylates BLNK inositol trisphosphate (IP3)

Activated Syk phosphorylates

Guanine-nucleotide exchange factors Ras and Rac activate the MAP
(GEFS) that in turn activate small GTP kinase cascade
binding proteins Ras and Rac
 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:

• The mitogen-activated protein kinase (MAP kinase) pathway

• Increased in intracellular Ca2+ mediated by IP3
• 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-γ, 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 NFκB

The activated transcription factors AP-1, NFAT and NFκB induce B cell
proliferation, differentiation and effector mechanisms
 Differentiation in the periphery

YY
YY

YY B
Y
B
YY
YY

Y
B
YY
Y Y YY
YY
Y YY
Mature peripheral B cell recognises Ig-secreting plasma cell
B cell non-self antigen
in periphery
 Plasma cells

Surface Surface High rate Growth Somatic Isotype

Ig MHC II Ig secretion hypermut’n switch

B High Yes No Yes Yes Yes

Mature B cell

B
Low No Yes No No
Plasma cell No
 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
B cells in
blood
T cell area

B cell
area

Efferent
lymph
 Recirculating B cells are trapped by foreign
antigens in lymphoid organs
B cells leave blood &
enter lymph node via
B cells high endothelial venules
proliferate
rapidly

Antigen enters
node in afferent
lymphatic Y
YY
Y
YYY
YY

Y
Y
YYY
Y Germinal centre
YY
Y releases B cells
GERMINAL CENTRE that differentiate
Transient structure of into plasma cells
Intense proliferation
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 Iccosomes (black coated particles)
then called ICCOSOMES bind to and are taken up by B cell
surface immunoglobulin
 Uptake of Iccosomes/Antigen by B cells

Anti- B cell
Iccosomes bearing
different antigens

Y
Y Y B
Surface Ig captures antigen CD40
Cross-linking of antigen receptor activates B cell
Activated B cell expresses CD40
 Fate of Antigens Internalised by B cells

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

2. Binding and internalisation via

Ig induces expression
of CD40

B
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

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

Cytokines
IL-4
IL-5

B Th IL-6
IFN-γ
YYY TGF-β

Cytokines
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 Signal 2, and thus T cells,
by the B cell also
regulate which B cells are
Th activates
YB hypermutation in the clonally selected.
Signal 1 CDR - encoding
parts of the Ig genes Low affinity Ig takes up and
presents Ag to T cells
Day 6 Day 8 Day 12 Day 18 inefficiently.
Clone 1
Clone 2 Inefficient presentation to T
Clone 3 cells does not induce CD40.
Clone 4
Clone 5
Clone 6 With no signal 2 delivered
Clone 7 by CD40, low affinity B cells
Clone 8 die.
Clone 9
Clone 10
Only B cells with high affinity
CDR2

CDR2

CDR3

CDR2

CDR3
CDR1

CDR3

CDR1

CDR1

CDR1
CDR2

CDR3

Ig survive - This is affinity

Deleterious mutation Lower affinity - Not clonally selected maturation
Beneficial mutation Higher affinity - Clonally selected
Neutral mutation Identical affinity - No influence on clonal selection
 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
B B B B B

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
B
Cytokines B B
B
IL-4
B PC
B Th IL-5
IL-6
B B B
YYY B
IFN-γ B B
TGF-β B
B B

Signal 1 Proliferation & Differentiation

IgM IgG3 IgG1 IgG2b IgG2a IgE IgA

IL4 inhibits inhibits induces inhibits induces

IL-5 augments
IFN-γinhibits induces inhibits induces inhibits
TGF-β 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
B cells leave blood &
enter lymph node via
B cells high endothelial venules
Rapidly
proliferate
in follicles
Antigen enters
node in afferent
lymphatic Y
YY
Y
YYY
YY

Y
Y
YYY
Y Germinal centre
YY
Y releases B cells
GERMINAL CENTRE that differentiate
Transient structure of into plasma cells
Intense proliferation
 1. Antigen loaded dendritic cells
migrate from subcapsular
sinus to paracortical area
B cells (90%) and T cells
of the lymph node
(10%) migrate to form
a primary follicle
Primary follicle
B B
formation
B T 3. T cells
B B proliferate T
B DC
2. T cells migrate
B B through HEV and
T
B T T are trapped by
B T T T antigen on DC
B B T
4. B cells migrate through
HEV - most pass through the B
paracortex and primary follicle. HEV
Some interact with T cells and
proliferate to form a primary focus
T cell motility in the lymph node
 Germinal Centre Microanatomy
2. B cells (centrocytes) upregulate
surface Ig, stop dividing and 4. Selected cells leave lymph
receive costimulatory signals node as memory
from T cells and FDC cells or plasma cells

Primary Follicles become

Light zone
secondary
B follicles 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

B B PC
B2 B cells

Y
?
YYYY
Y
Y
YY IgG
CD5

?
B B Y
Y B1 B cells
Y
Y Y

‘Primitive’ B cells found in

Y
Y Y
Y
Y
Y

pleura and peritoneum

Y
Y
Y
Y

Distinct B cell
Y
Y
Y
Y Y

precursor
IgM - no other isotypes
 B-1 B Cells

IgM uses a distinctive & restricted range of V regions

CD5
Few non-template encoded (N) regions in the IgM
Recognises repeating epitope Ag such as
B Y
Y
Y
Y Y

phospholipid phosphotidyl choline &

polysaccharides
Y
Y Y
Y NATURAL ANTIBODY
Y
YY
Y
Y
Y

Y
Y
Y
Y
Y

NOT part of adaptive immune response:

IgM
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 B-1 cells B-2 cells

N regions Few Extensive
V region repertoire Restricted Diverse
Location Peritoneum/pleura Everywhere
Renewal Self renewal in situ Bone marrow
Spontaneous Ig production High Low
Isotypes IgM IgM/G/A/D/E
Carbohydrate specificity
Carbohydrate specificity Yes
Yes Rarely
Protein specificity
Protein specificity Rarely
Rarely Yes
Need T cell
cell help
help No
No Yes
Somatic hypermutation of Ig No High
Memory development No 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)
TI-2 Antigen

Y
Immature
B-2 Cell

Y
YY Mature

Y
B-1

Y
Y
Y Y

Y
Y Y
Y
Y Y
Y

Y
Y Y
Y
Y Y
Immature B cells that bind to
Y
Y

multivalent self Ag undergo
apoptosis
B-2 cell repertoire is purged
of cells recognising
multivalent antigens during
development in the bone
marrow
Y
Y Y
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 Bacterial Lipopolysaccharides, (TI-1

protein antigens), bind to host LPS binding
protein in plasma
LPS
TLR 4
LPS/LPSBP is captured by CD14
on the B cell surface

Toll - like receptor 4 (TLR4) interacts

with the CD14/LPS/LPSBP complex

CD14

Activation of B cell
B Cell
 T Independent Antigens (TI-1 e.g. LPS)
LPS complexes with CD14, LPSBP & TLR4

B B B B B B
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
YY YY YY YY YY YY
YY YY YY YY YY YY
Y YY YY YY YY YY Y
 T Dependent & Independent Antigens
T
Dependent TI-1 TI-2
Antigens Antigens Antigens
Induce responses in babies Yes Yes No
Induce responses in athymics No Yes Yes
Prime T cells Yes No No
Polyclonally activate B cells No Yes No
Require repeating epitopes No No Yes
Why are babies unresponsive to TI-2 antigens?
In adults: TI-2 Antigen

Y
Immature
B-2 Cell

Y
YY Mature

Y
B-1

Y
Y
Y Y

Y
Y Y
Y
Y Y
Y

Y
Y Y
Y
Y Y
Adult immature B cells that
Y
Y

Y
YY
bind to multivalent self Ag
undergo apoptosis 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
YY
Immature B cells that bind to
multivalent self Ag undergo
apoptosis
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
T
Dependent TI-1 TI-2
Antigens Antigens Antigens
Induces response in babies Yes Yes No
Induces response in athymia No Yes Yes
Primes T cells Yes No No
Polyclonally activates B cells No Yes No
Requires repeating epitopes 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
Toxin

Y
Bacterium

`
Y
`

`
` `
Adhesion to
host cells blocked
Toxin release
blocked
Y
`
Y
`

Prevents Prevents
invasion toxicity

NEUTRALISING ANTIBODIES
 Effector mechanisms against
extracellular pathogens
OPSONISATION
Bacteria in extracellular space

+
Ab

OPSONISATION Fc receptor Phagocytosis

binding
 Effector mechanisms against
extracellular pathogens
COMPLEMENT Activation
Bacteria in plasma
Lysis

+
Ab &
COMPLEMENT

Opsonisation Complement & Phagocytosis

Fc receptor
binding
 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