B Cells and B Cell Development

© Dr. Colin R.A. Hewitt

crah1@le.ac.uk

© crah1@le.ac.uk 2000. Slide 1/33

 This is second in the series of cellular immunology lectures.
The purpose of the lecture is to illustrate the key points of B cell
biology and to emphasise the mechanisms that B cells use to
generate a diverse B cell repertoire whilst preventing self reactivity.

To use the lecture, click on the projection screen icon below

then just click your way through the presentation

Don’t forget to try the online multiple choice questions at the end to find your
strengths and weaknesses.

© crah1@le.ac.uk 2000. Slide 2/33

What you should know by the end of this lecture

• B cells develop pre-natally in the liver, and post-natally in the

bone marrow
• Anti-self B cells are eliminated once Ig is expressed at the cell
surface
• Self-reactivity is controlled by clonal deletion and clonal anergy
• Stages of B cell differentiation are defined by Ig gene
rearrangement
• The structure and function of the pre-B cell receptor
• The meaning of, purpose of and evidence for allelic exclusion
• How the order of Ig gene rearrangement makes B cell
development efficient
• The development and functions of the germinal centre

© crah1@le.ac.uk 2000. Slide 3/33

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

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
© crah1@le.ac.uk 2000. Slide 4/33
 Origin of B cells and organ of B cell
maturation

Mature B cells
Transfer foetal in periphery
liver cells
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

© crah1@le.ac.uk 2000. Slide 5/33

 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
© crah1@le.ac.uk 2000. Slide 6/33
 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
© crah1@le.ac.uk 2000. Slide 7/33
 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
© crah1@le.ac.uk 2000. Slide 8/33
 Cytokines and cell-cell contacts at each
stage of differentiation are different

Early Kit
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

© crah1@le.ac.uk 2000. Slide 9/33

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
© crah1@le.ac.uk 2000. Slide 10/33
 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

© crah1@le.ac.uk 2000. Slide 11/33
 Pre- B cell receptor
Heavy chain
VHDHJH

Light chain VpreB

VLJLCL
CHm l5
Iga & Igb signal
transduction
molecules

Transiently expressed when VHDHJH CHm is productively rearranged

VpreB/l5 - the surrogate light chain, is required for surface expression

Ligand for the pre-B cell receptor is unknown

© crah1@le.ac.uk 2000. Slide 12/33
 Ligation of the pre-B cell receptor

1. Suppresses further H chain rearrangement

2. Triggers entry into cell cycle

Large Unknown ligand of

Pre-B 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

© crah1@le.ac.uk 2000. Slide 13/33

 Evidence for allelic exclusion
ALLOTYPE- a polymorphism in the C region of Ig
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

© crah1@le.ac.uk 2000. Slide 14/33

Allelic exclusion prevents unwanted responses
One Ag receptor per cell IF there were two Ag receptors per cell

Y
B Self antigen
B
YY expressed by
e.g. brain cells
YY
S. aureus
Y S. aureus Y
Y Anti
Y
S. aureus
Anti
brain
Y Anti
Y
S. aureus

Y Antibodies Abs
Y Antibodies

Suppression of H chain gene rearrangement

ensures only one specificty of Ab expressed per cell.
Prevents induction of unwanted responses by pathogens
© crah1@le.ac.uk 2000. Slide 15/33
 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
© crah1@le.ac.uk 2000. Slide 16/33
Allelic exclusion is needed to prevent holes in the repertoire

One Ag receptor per cell IF there were two Ag receptor per cell

Anti-brain Ig Anti-brain Ig
AND
anti-S. Aureus Ig
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
S. aureus
B

© crah1@le.ac.uk 2000. Slide 17/33

 Ligation of the pre-B cell receptor

1. Suppresses further H chain rearrangement

2. Triggers entry into cell cycle

Large Unknown ligand of

Pre-B 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

© crah1@le.ac.uk 2000. Slide 18/33

 Large pre-B cells need in frame VHDHJH joins to mature
Human IgG3 Heavy Chain
nucleotide sequence
ATGAAACANCTGTGGTTCTTCCTTCTCCTGG
TGGCAGCTCCCAGATGGGTCCTGTCCCAGG
TGCACCTGCAGGAGTCGGGCCCAGGACTGG
GGAAGCCTCCAGAGCTCAAAACCCCACTTGG
TGACACAACTCACACATGCCCACGGTGCCCA
GAGCCCAAATCTTGTGACACACCTCCCCCGT
GCCCACGGTGCCCAGAGCCCAAATCTTGTG
ACACACCTCCCCCATGCCCACGGTGCCCAG
AGCCCAAATCTTGTGACACACCTCCCCCGTG
CCCNNNGTGCCCAGCACCTGAACTCTTGGG
AGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGATACCCTTATGATTTCCCGGACCC
CTGAGGTCACGTGCGTGGTGGTGGACGTGA
GCCACGAAGACCCNNNNGTCCAGTTCAAGT
GGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCTGCGGGAGGAGCAGTACA
ACAGCACGTTCCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAACGGCAA
GGAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGACAGCCCGAGGAGATGACCA
AGAACCAAGTCAGCCTGACCTGCCTGGTCAA
AGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAACACCACGCCTCCCATGCTGGACTCC
GACGGCTCCTTCTTCCTCTACAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACATCTTCTCATGCTCCGTGATGCATGAGGC
TCTGCACAACCGCTACACGCAGAAGAGCCTC
TCCCTGTCTCCGGGTAAATGA
Translation in frame 1 Development
continues
MKXLWFFLLLVAAPRWVLSQV
HLQESGPGLGKPPELKTPLGD
Large Pre-B cell
TTHTCPRCPEPKSCDTPPPCP
RCPEPKSCDTPPPCPRCPEPK
pre-B receptor can
SCDTPPPCXXCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTC ligate to
VVVDVSHEDXXVQFKWYVDG stromal cell
VEVHNAKTKLREEQYNSTFRV
VSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPEE
MTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYNTTPPMLD
SDGSFFLYSKLTVDKSRWQQG
NIFSCSVMHEALHNRYTQKSLS
LSPGK*
Development
Translation in frame 2
(no protein) arrests
*
Translation in frame 3 Development
ETXVVLPSPGGSSQMGPVPGA
PAGVGPRTGEASRAQNPTW*
arrests
© crah1@le.ac.uk 2000. Slide 19/33
 Ligation of the pre-B cell receptor triggers
entry into the cell cycle

Large Large
Pre-B Pre-B
Large
Large
Large
Large
Many large pre-B
Large Proliferation Pre-B
Large
Pre-B
Pre-B
Large
Pre-B cells with identical
Large
Pre-B Large
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
© crah1@le.ac.uk 2000. Slide 20/33
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
© crah1@le.ac.uk 2000. Slide 21/33
 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 k on first
YES YES IgMk
On first On first chromosome
chromosome
NO
YES
chromosome

NO YES
NO
k on second
chromosome
YES
Y
B
DH-JH VH-DJH NO
On second On second
l on first YES
chromosome chromosome
chromosome IgMl
NO
NO
NO
l on second
chromosome
YES
Y
B
B
NO

© crah1@le.ac.uk 2000. Slide 22/33

 Acquisition of antigen specificity creates a
need
to check for recognition of self antigens
Y

Y
Y
B B
Y
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 DELETION

2. Paralysis of function ANERGY
3. Alteration of specificity RECEPTOR EDITING
© crah1@le.ac.uk 2000. Slide 23/33
 B cell self tolerance: clonal deletion

Small
B
B
pre-B Immature

YY B B

Small pre-B cell Immature Clonal deletion by

assembles Ig B cell recognises apoptosis
MULTIVALENT
self Ag

© crah1@le.ac.uk 2000. Slide 24/33

 B cell self tolerance: anergy

IgD normal IgM low

Small
YY IgM

B
pre-B Immature IgD
B IgD
B B
IgD

B
Small pre-B cell Immature
assembles Ig B cell recognises
soluble self Ag
No cross-linking
Anergic B cell
© crah1@le.ac.uk 2000. Slide 25/33
 Receptor editing
A rearrangement encoding a self specific receptor can be replaced

V V V V D J C

!!Receptor
recognises
B 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

B a different antigen and can be
rechecked for specificity
© crah1@le.ac.uk 2000. Slide 26/33
B cell self tolerance: export of self tolerant B cells

IgD and IgM normal

Small
YY IgM
IgD
IgM
B
pre-B Immature
BB IgD IgD
B
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
© crah1@le.ac.uk 2000. Slide 27/33
 Differentiation in the periphery

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

© crah1@le.ac.uk 2000. Slide 28/33

 Recirculating B cells normally pass through
lymphoid organs
B cells in
blood
T cell area

B cell
area

Efferent
lymph

© crah1@le.ac.uk 2000. Slide 29/33

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

Antigen enters
node in afferent
lymphatic Y
YY

Y
YY

Y
Y
Y
Germinal centre
YY
Y releases B cells
GERMINAL CENTRE that differentiate
Transient structure of into plasma cells
Intense proliferation
© crah1@le.ac.uk 2000. Slide 30/33
 Germinal centre anatomy
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

Light zone B Dark zone

T

Follicular dendritic
cells select useful
B cells 1. B cells (centroblasts) downregulate
3. Apoptosis of surface Ig, proliferate, somatically
self-reactive & hypermutate their Ig genes.
unselected cells AFFINITY MATURATION
© crah1@le.ac.uk 2000. Slide 31/33
 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
• Anti-self B cells are eliminated by clonal deletion and anergy
• Mature B cells develop in germinal centres

NOW TRY THE MULTIPLE CHOICE QUESTIONS

(click on this link)

© crah1@le.ac.uk 2000. Slide 32/33

 Further Reading:
B cells and B cell Development
N.B. You are not expected to read all of these references, but they should
point you to specific areas that are covered in the lecture that you need
more information on.

• Goodenow, CC. et al., Self tolerance checkpoints in B cell development Adv.

Immunol. 1995 59:279-368
• Kelsoe G. In situ studies of the germinal centre reaction Adv. Immunol. 1995
60:267-288
• Winkler T. and Rolnik, AG. Roles of Ig H-chain and L-chain and of surrogate H-
chain and L-chain in the development of cells of the B lymphocyte lineage. Ann.
Rev. Immunol. 1994 12:209-225
• Schatz, DG. et al.,V(D)J recombination - molecular biology and regulation.
Ann. Rev. Immunol. 10:359-383
• Hartley, SB. et al.,Elimination of self-reactive B lymphocytes proceeds in two
stages – arrested development and cell death. Cell. 1993 72:325-335
• Jacob J. et al., Intraclonal generation of antibody mutants in germinal centres.
Nature. 1991 354:389-392
• Maclennan ICM. Germinal Centres. Ann. Rev. Immunol. 1994 12:117-139
• Chen C. et al., The site and stage of anti-DNA B cell deletion. Nature. 1995
373:252-255
© crah1@le.ac.uk 2000. Slide 33/33