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 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
CH

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

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

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

Y
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

IgD

Immature
B

IgD

Small
pre-B

YY

Y 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

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

IgD

YY

IgM

YY
B

YY

Immature
B

YY

Small
pre-B

YY

IgD and IgM normal

IgD
IgM
IgM
IgD

IgM

IgD

Small pre-B cell

assembles Ig

Immature
B cell doesnt
recognise any
self Ag

Mature B cell
exported to the
periphery

Splicing of IgM and IgD RNA

VD J

VD J

C1

C2

C3

C3

C1

C4

C1

C2

DNA

C3

pA2

pA1

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?

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

YY
Y

YY

YY
Mature peripheral
B cell

B cell recognises
non-self antigen
in periphery

Y
Y
Y

YY
YY
YY

YY

YY
Y

YY

Differentiation in the periphery

Ig-secreting plasma cell

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

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

Y
B

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

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

PC

B
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

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

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

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
Y

NATURAL ANTIBODY

Recognises repeating epitope Ag such as

phospholipid phosphotidyl choline & polysaccharides

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

IgM

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
Proteinspecificity
specificity
Need T cell
cell help
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
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)

Immature
B-2 Cell

Y
Y
Y

Y
Mature
B-1

Y
Y

Y
Y

Y
Y

YY

Y
Y

Y
Y

Y
Y

B-2 cell repertoire is purged

of cells recognising
multivalent antigens during
development in the bone
marrow

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 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 to 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

Immune effector mechanisms against

extracellular pathogens & toxins
NEUTRALISATION
Toxin

Bacterium

`
`

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
There are two lineages of B cells - B1 and B2 B cells
The dependency of B cells upon T cells varies