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B Cell repertoire - role of B cell antigen receptors (BCR)

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To generate enough antibodies for a large number of pathogens, adaptive immunity could have evolvedby producing one gene for each antibody. Disadvantages/problems:

- how does the body know of all the pathogens that they would encounter?

But, our immune system generates a random set of antibodies by shuffling a relatively small number of genes.

- waste of resources

- would need a large genome

- do not provide the immune system with the flexibility of fighting new infections in a specific way

- generation of antibodies against self-antigens

Disadvantages/problems:

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B cell development

Ig heavychain

VpreBplus 5

Ig lightchain

proB preB I ImmatureBHeavy chain

generearrangement

preB IILight chain

generearrangement

matureB

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Generation of a diverse repertoire of immunoglobulins (Ig) (1) - heavy chain gene

VH segments JH segmentsD segments

Recombination ofD and JH segments

Recombination ofV and DJH segments

Constantregion

5

V segments J segments

Recombination ofV and J segments

Generation of a diverse repertoire of immunoglobulins (Ig) (2) - light chain gene

Constant region

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Generation of a diverse repertoire of antibodies

(1) Random rearrangement of variable gene segments

(2) Different combinations of heavy (IgH) and light chains (Ig or Ig) (3) Addition or deletion of nucleotides at the junction where the variable gene segments are joined

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Proteins:

VpreB + 5 = surrogate light chain - part of pre-BCR

RAG-1 + RAG-2 = recombinase

nucleases = nick and digest hairpin loops of intermediate

TdT (terminal deoxynucleotide transferase) = for N-addition

IgH, Ig, Ig = containing variable regions for recognition of antigens

Ig, Ig = (1) for cell surface expression of immunoglobulin; (2) for signaling

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proB preB IImmature

BpreB IImature

B

positive selection of B cells -BCR signals promote proliferation and differentiation

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5 and VpreB KO mice- Delayed B cell development, but not abolished - normal numbersof peripheral B cells 6 months later- No apparent defect in allelic exclusion

Role of preB receptor:- suppress RAG genes expression, such that allelic exclusion of IgH isachieved- promote proliferation of preB cells to finish rearrangements of IgL

Function of 5 and VpreB - may select for heavy chain variable regions - shaping the primary B cell repertoire

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Antigen-dependent maturation of B cells:

- Fix IgH chain expression in transgenic mice- These mice utilize endogenous light chains- Isolated immature and mature B cells separately -> measure theusage of light chain variable gene segments

Hypothesis:If maturation of immature B cells (to mature B cells) is random, antibody repertoire remains unchanged

Result:Repertoire significantly changed

Conclusion:Maturation of immature B cells is not random, suggesting antigendependent

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V-A V-B V-C V-A V-B V-C

Stochasticevents

Antigen-dependentselection

Testing dependence on antigen selection during maturation of B cells

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Activation of B cell antigen receptor (BCR) signaling

YY

BCR

Syk

-p-p

-p-p

p

p

p

p

SLP65Bruton tyrosine kinasePLC-

YY

YY

antigen

Lyn/Fyn/Blk

Proliferation

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Ligand-independent signaling through (pre-) B cell BCR

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-p-p

p

p

YY

proB preB IImmature& mature

B

preB II

Normal mouse

MT mouse

CD22- CD43+

CD22+ CD43-

MHC II+CD23+

CD22- CD43+

X X

CD22+ CD43-

MHC II+CD23+

CD22- CD43+

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-p-p

p

p

YY

-p-p

p

p

YY

-p-p

p

p

YY

-p-p

p

p

YY

Strong signal Weak maintenance signal

Antigen-dependent and independent (pre-) BCR signals

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proB preB IImmature

BpreB IImature

B

5MTSyk

BLNKBtkVavCD45Lyn

Regulation of positive selection by signaling pathways

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proB preB IImmature

BpreB IImature

B

negative selection of B cells -BCR signals induces death or anergy

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Activated B

deletion

B cell tolerance

Self-reactiveImmature B

deletion

Rescued

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Receptor editing by secondary rearrangement

V segments J segments

Recombination ofV and J segments

Constant region

SecondaryRecombination

(1)

(2) Rearrangement of the germline allele

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Receptor editing:

(1) Abnormally high or low levels of receptor signals stimulate receptor editing

(2) Interleukin-4, in combination with LPS or anti-CD40, stimulate expression of RAG-1 and -2 genes

(3) Interleukin-7, in combination with anti-CD40, stimulate RAG-1 and -2 genes expression

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Receptor editing (continued):

(4) c-myb and Pax-5 proteins activate RAG-2 gene expression

(5) E2A activate RAG-1 and RAG-2 genes expression

(6) OcaB, a transcription factor, promotes Ig gene recombination through the activation of germline gene transcription

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-p-p

p

p

YY

-p-p

p

p

YY

-p-p

p

p

YY

Strong signalleads to apoptosis

Weak signal leads to anergy

Deletion and anergy as mechanisms of immune tolerance -IgHEL/HEL mouse model

HEL

-p-p

p

p

YY

IgHEL

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Self-reactivity Shapes the B cell repertoire

AnergyMature B cell

Failure to enterthe periphery

B cell fate

Deletion

Strength of BCR signal

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IgM

preB IImmature

BpreB IImature

B

Antigen-inducedactivation

proB

IgG

Expression of Ig isotypes in normal B cells

MT

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Discussion of Seagal et al, 2003, J. Exp. Med.

Previous observation:

Hypothesis/Proposal:

Conclusion:

Results:

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Staining for cell surface maker

B CELL

PE

FITC

PE

FITC

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7