B CELL DEVELOPMENT IN THE BONE MARROW

OORDERED RDERED BB-CELL DEVELOPMENT-CELL DEVELOPMENT

pre B cellpro B cell

immature B cell

NO ANTIGEN RECOGNIZING RECEPTOR

H-chain + surrogate L-chainSIGNALING RECEPTOR

ANTIGEN RECOGNIZING RECEPTOR

H2L2

B

B

Stromal cell

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

Early pro-B

c-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)

D-J rearranged

No IgDJ rearrangement (H chain)L-chain genes in germline form

Interleukin-7receptor

Stromal cell

Late pro-B Pre-B

Interleukin-7Growth factor

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

Early pro-B

VLA-4(Integrin)

VCAM-1(Ig superfamily)

V-D-J rearrangedLight chain germline

µ-chain made

B cell receptor

Transiently expressed when VHDHJH CH is productively rearranged

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

Ig & Ig signaltransductionmolecules

CH

Heavy chainVHDHJH

V-preB

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

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-J light chainrearranged V-J light chain expression is quite

efficient with an 85% success rateFrom a single µ chain 85 receptor!!!

100X expansion

B cell receptor

Ig & Ig signaltransductionmolecules

CH

Heavy chainVHDHJH

Light chainVLJLCL

L chain is rearranged

RECEPTOR EXPRESSION DURING B-CELL DEVELOPMENT

even though every B cell possesses a maternal and paternal locus of both genes, B cells express a single heavy and light chain. Does this „crippled” expression serve a purpose? Many of the genes (not all) are expressed co-dominantly, how could B cells manage to silence their other BCR-coding allels?

ALLELIC EXCLUSION

Allelic exclusion

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 Heavy chain C region of Ig

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

specificities of antibody per cell

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 to prevent the emergence ofnew daughter specificities during proliferation after clonal selection

S. typhi

Antibody

S. typhi

YY

Y Y

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

Allelic exclusion prevents unwanted responses

BSelf antigenexpressed by

e.g. Liver cells

S. aureusY Y

YYYB

S. aureus

YY

Y

YY

Y

Y

AntiS. aureus

Antibodies

Y

Y Y

Y Y

YAntiLiver cell

Abs

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 to prevent holes in the repertoire

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

YYBB

One specificity of Agreceptor per cell

S. aureus

Anti-brain IgAND

anti-S. Aureus IgYYYBB

IF there were two specificitiesof 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. Combination of gene segments results in a huge number of various variable regions of the heavy and light chains expressed by different B-cells

SOMATIC GENE REARRANGEMENT

2. Successful somatic rearrangement in one chromosome inhibits gene rearrangement in the other chromosome

ALLELIC EXCLUSION

3. One B-cell produces only one type of heavy and one type of light chain

COMMITMENT TO ONE TYPE OF ANTIGEN BINDING SITE

4. The B-cell pool consist of B-cells with differently rearranged immunoglobulin genes

THE RESULT OF SOMATIC GENE REARRANGEMENTS

INDEPENDENT OF ANTIGEN

OCCURS DURING B-CELL DEVELOPMENT IN THE BONE MARROW

Allelic exclusion helps diagnose and monitor lymphoma:

Due to clonal expansion of a single cell that contains a unique rearrangement the amount of cancer cells in blood or in bone

marrow can be determined

Can be used to monitor residual tumor cells upon treatment

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 IgH and IgL chain genes, expression of adhesion molecules and cytokine receptors

Y

ReceptorH+L

YReceptor

H+L

SYNTHESIS OF IMMUNOGLOBULINS

ER

Golgi

mRNA

Ribosome

Leader sequence

Membrane Ig Secreted Ig

H and L chains are synthesized on separated

ribosomes

CHAPERONES

B

B

B

B

c-kit/CD44

RAG-1/RAG-2

Limphoid precursor

H átrendeződésH rearrrangement

Surrogate L

L rearrangement

Selectionclonal deletion

DEVELOPMENT OF B-LYMPHOCYTES IN THE BONE MARROW

PERIPHERAL LYMPHOID TISSUES

B cells recognizing self structures

Cell surface moleculesMHC proteinsCommon molecules of haemopoetic cells

apoptosis, clonal deletion

Soluble moleculesHouse keeping genesMetabolites

functional unresponsiveness

anergia

Other specificites

n3 42 51BONE

MARROW

Potential B-cell repertoire

Self structure

Self recognitionClonal deletion

PERIPHERAL LYMPHOID ORGANS

Available B-cell repertoireForeign antigen independent

About 30 billion mature naive B cells leave the bone marrow per day to circulate in blood

Negative selection of immatureB-cells in the bone marrow

RNA editing

Immature B cells with specificity for multivalent self antigens are retained in the bone marrow.

Receptor Editing of Immature B cells with self-reactive BCR (Bone Marrow)

Immature B cells specific for monovalent self antigens develop a state of anergy.

Anergic B cells have a halflife of 4-5 days (10% that of regular B cells)

How can mature B-cells express surface IgM and IgD

Co-Expression of cell surface IgM and IgGOn Mature B-cells is controlled by alternative

RNA processing

1. Somatic rearrangement of Ig gene segments occurs in a highly controlled manner

2. Single B-cells become committed to the synthesis of one unique H-chain and one unique L-chain variable domain, which determine their specificities

3. In one individual a large B-cell repertoire is generated consisting of B-cell clones with different H- and L-chain variable domains

4. This potential B-cell repertoire is able to recognize a wide array of various antigens

5. Immature B-cells express IgM and IgD surface Ig with the same variable domains

RESULT OF SOMATIC GENE REARRANGEMENT AND ALLELIC EXCLUSION

B – CELL ACTIVATION

Where and how do all these things take place?

B-cell recycling in the absence of antigen (lymph node)

B cells in blood

Efferenslymph

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 leave blood & enter lymph node via

high endothelial venulesB 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

Germinal Center Reaction

IV_10_5_Germinal_Centers-H264.mov

„Dating” in the peripheral lymphoid organs

The structure of the germinal centre

Somatic hypermutation

FDC

Somatic hypermutation

DZ

LZ

LZ: light zoneDZ: dark zoneFDC: follicular dendritic cell

Antigen is bound on the surface of follicular dendritic cells (FDC)

FDC FDC-s bind immune complexes (Ag-Ab ) Ag detectable for 12 months following immunization A single cell binds various antigens

B cells recognize Ag on the surface of FDC

Fig. 9.15. On the surface of FDC-s immune complexes form the so-called iccosomes,that can be released and taken up later by the surrounding germinal center B cells

T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID ORGANS

IgM

IgGIgAIgE