© [email protected] 2000. [email protected] slide 1/33 b cells and b cell development © dr. colin r.a....

© [email protected] 2000. Slide 1/33

B Cells and B Cell Development

© Dr. Colin R.A. Hewitt

[email protected]


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.


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


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 apparenteffect

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


Transfer foetalliver cells

Origin of B cells and organ of B cellmaturation

No MatureB cells

After birth, development continues in the bone marrow

Normal bone marrow

Defective bone marrow

Mature B cellsin periphery

B cell development starts in the foetal liver


B cell development in the bone marrow

B Regulates construction of an antigen receptor

Bone Marrow provides aMATURATION & DIFFERENTIATION MICROENVIRONMENT

for B cell development

Ensures each cell has only one specificityB

Checks and disposes of self-reactive B cellsB

Exports useful cells to the peripheryB

Provides a site for antibody productionB


SecretedFactors - CYTOKINES

2. Secretion of cytokines by stromal cells

B

Bone marrow stromal cells nurture developing B cells

Types of cytokines and cell-cell contacts needed at each stage of differentiation are different

Stromal cell

1. Specific cell-cell contacts between stromal cells and developing B cells

Cell-cell contact


Peripheral

Stages of B cell development

Stem Cell Early pro-B cell Late pro-B cell Large pre-B cell

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


Early pro-B

KitReceptor Tyrosinekinase

Stem cell factor

Cell-bound growthfactor

VLA-4(Integrin)

Stem

Cytokines and cell-cell contacts at each stage of differentiation are different

Stromal cell

Cell adhesionmolecules

VCAM-1(Ig superfamily)


Early pro-B

Interleukin-7receptor

Stromal cell

Late pro-B Pre-B

Interleukin-7Growth factor

Cytokines and cell-cell contacts at each stage of differentiation are different


Stages of differentiation in the bone marrow aredefined by Ig gene rearrangement

B CELL STAGE

IgH GENECONFIGURATION

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

Germline DH to JH VH to DHJH VHDHJH

Pre-B cellreceptor

expressed

Ig Light chain gene has not yet rearranged


B cell receptor

Transiently expressed when VHDHJH CH is productively rearranged

VpreB/5 - the surrogate light chain, is required for surface expression

Ig & Ig signaltransductionmolecules

CH

Heavy chainVHDHJH

Light chainVLJLCL

VpreB

5

Ligand for the pre-B cell receptor is unknown

Pre-


Ligation of the pre-B cell receptor

1. Ensures only one specificty ofAb expressed per cell

LargePre-B

Stromal cell

Unknown ligand of pre-B cell receptor

2. Triggers entry into cell cycle

ALLELIC EXCLUSION

1. Suppresses further H chain rearrangement

2. Expands only the pre-Bcells with in frame VHDHJH joins


Evidence for allelic exclusion

Allotypes can be identified by staining B cell surface Ig with antibodies

a/a b/b a/b

YBb YBa YBb

Y

YB ab

YBa AND

ALLOTYPE- a polymorphism in the C region of Ig

Suppression of H chain rearrangement by pre-B cell receptor prevents expression of two

specificities of antibody per cell


YY

Y Y

Suppression of H chain gene rearrangementensures only one specificty of Ab expressed per cell.

Allelic exclusion prevents unwanted responses

BSelf antigenexpressed bye.g. brain cells

S. aureusY Y

YYYB

S. aureus

YY

Y

YY

Y

Y

AntiS. aureus

Antibodies

Y

Y Y

Y Y

YAntibrainAbs

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

Y

Y Y

Y

YY Y

AntiS. aureus

Antibodies

Prevents induction of unwanted responses by pathogens


Allelic exclusion is needed for efficient clonal selection

All daughter cells must express the same Ig specificityotherwise the efficiency of the response would be compromised

Suppression of H chain gene rearrangement helps prevent the emergence ofnew daughter specificities during proliferation after clonal selection

S. typhi

Antibody

S. typhi


Allelic exclusion is needed to prevent holes in the repertoire

Exclusion of anti-brain B cells i.e. self tolerance

YYBB

One Ag receptor per cell

S. aureus

Anti-brain IgAND

anti-S. Aureus IgYYYBB

IF there were two Ag receptor per cell

Anti-brain Ig

BB

Deletion Anergy

OR

anti S.Aureus B cells will be excluded leaving a “hole in the repertoire”

BUT

YYYBB


1. Suppresses further H chain rearrangement

Ligation of the pre-B cell receptor

1. Ensures only one specificty ofAb expressed per cell

LargePre-B

Stromal cell

Unknown ligand of pre-B cell receptor

2. Triggers entry into cell cycle

2. Expands only the pre-Bcells with in frame VHDHJH joins


Translation in frame 1

MKXLWFFLLLVAAPRWVLSQVHLQESGPGLGKPPELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCXXCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDXXVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRYTQKSLSLSPGK*

Human IgG3 Heavy Chain nucleotide sequence

ATGAAACANCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCACCTGCAGGAGTCGGGCCCAGGACTGGGGAAGCCTCCAGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCNNNGTGCCCAGCACCTGAACTCTTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCNNNNGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCGAGGAGATGACCAAGAACCAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTACACGCAGAAGAGCCTCTCC

CTGTCTCCGGGTAAATGA

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

Translation in frame 2(no protein)

*

Translation in frame 3

ETXVVLPSPGGSSQMGPVPGAPAGVGPRTGEASRAQNPTW*

Largepre-B

Development continues

Pre-B cell receptor can

ligate to stromal cell

Developmentarrests

Developmentarrests


LargePre-B

LargePre-B

LargePre-B

LargePre-B Large

Pre-BLargePre-B Large

Pre-BLargePre-B Large

Pre-BLargePre-B

Proliferation

Y ImmatureB cell

Light chain expressedIgM displayed on surface

IgM

Ligation of the pre-B cell receptor triggers entry into the cell cycle

Largepre-B

Many large pre-B cells with identical pre-B receptors

Large pre-B

Intracellular VDJCH chainVL-JL rearranges

Proliferation stops

Pre-receptor not displayed

Small pre-B


V D J C

D JV CD J

V CD JV

Germline

DH-JH joining

VH-DHJH joining

V J C

V CJV

Germline

VL-JL joining

Heavy and light chain rearrangement is potentially wasteful

Largepre-B

Smallpre-B

With two “random” joins to generate a heavy chainthere is a 1:9 chance of a rearrangement of being in frame

With one “random” join to generate a light chainthere is a 1:3 chance of a rearrangement being of frame

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 successfullyrearrange Ig genes

Early Pro B Late Pro B Pre B

VH-DJH

On firstchromosome

VH-DJH

On secondchromosome

Immature B

NO

YES

NO

B

on firstchromosome

on secondchromosome

on firstchromosome

on secondchromosome

NO

NO

NO

NO

YES

YES

NO

YES

YES

YES

YES

YIgM

B

YIgM

B

DH-JH

On firstchromosome

DH-JH

On secondchromosome

YESNO


B Y

Y

Y

YB

Small pre-B cell

No antigen receptor at cell surfaceUnable to sense Ag environment

!!May be self-reactive!!

Immature B cell

Cell surface Ig expressedAble to sense Ag environment

Can now be checked for self-reactivity

Acquisition of antigen specificity creates a need

to check for recognition of self antigens

1. Physical removal from the repertoire DELETION2. Paralysis of function ANERGY3. Alteration of specificity RECEPTOR EDITING


B cell self tolerance: clonal deletion

Immature B cell recognisesMULTIVALENT

self Ag

B

Clonal deletion byapoptosis

YYBImmature

BB

Smallpre-B

Small pre-B cellassembles Ig

© [email protected] 2000. Slide 25/33Y

B cell self tolerance: anergy

B

YY

YB

Anergic B cell

IgD normal IgM low

Immature B cell recognisessoluble self Ag

No cross-linking

YYB

ImmatureB

BSmallpre-B


IgM

IgD

IgD

IgD


Receptor editingA rearrangement encoding a self specific receptor can be replaced

V CD JVV V

Y

BB!!Receptorrecognises

self antigen!!

B Apoptosisor anergy

YBB

Edited receptor now recognisesa different antigen and can be

rechecked for specificity

CD JVV VV

Arrest developmentAnd reactivate

RAG-1 and RAG-2


YY

Y

YYY

Mature B cellexported to the

periphery

Y

Y

B cell self tolerance: export of self tolerant B cells

IgD and IgM normal

IgMIgD

IgD

IgD

IgD

IgMIgM

IgM

Immature B cell doesn’t recognise any

self Ag

YYB

ImmatureB

BSmallpre-B


B


B cell recognisesnon-self antigen

in periphery

Ig-secreting plasma cell

Differentiation in the periphery

YY Y YY YYY Y

B

Y Y

Y

Y

YYY

BY Y

YY

YYYB

Mature peripheralB cell


Recirculating B cells normally pass throughlymphoid organs

B cells inblood

Efferentlymph

T cell area

B cell area


Antigen entersnode in afferent

lymphatic

Y

Y

Y

Y

Y

YY

Y

Y

Y

Y

Y

Y

Y

YY

Y

YB cells enter lymph node

and leave blood viahigh endothelial venules

B cellsproliferate

rapidly

GERMINAL CENTRETransient structure ofIntense proliferation

Germinal centrereleases B cellsthat differentiateinto plasma cells

Recirculating B cells are trapped by foreign antigens in lymphoid organs


Dark zoneLight zone B

T

Follicular dendriticcells select useful

B cells

Germinal centre anatomy

1. B cells (centroblasts) downregulatesurface Ig, proliferate, somatically

hypermutate their Ig genes.AFFINITY MATURATION

2. B cells (centrocytes) upregulatesurface Ig, stop dividing andreceive costimulatory signals

from T cells and FDC

3. Apoptosis ofself-reactive &

unselected cells

4. Selected cells leave lymphnode as memory

cells or plasma cells


• 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

Summary:

NOW TRY THE MULTIPLE CHOICE QUESTIONS(click on this link)


Further Reading:Further Reading:B cells and B cell DevelopmentB 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

© [email protected] 2000. [email protected] slide 1/33 b cells and b cell development © dr. colin r.a....

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