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B CELL DEVELOPMENT IN THE BONE MARROW
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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
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B
B
Stromal cell
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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
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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
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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
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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-
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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
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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
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B cell receptor
Ig & Ig signaltransductionmolecules
CH
Heavy chainVHDHJH
Light chainVLJLCL
L chain is rearranged
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RECEPTOR EXPRESSION DURING B-CELL DEVELOPMENT
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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
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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
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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
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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
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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
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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
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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
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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
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SYNTHESIS OF IMMUNOGLOBULINS
ER
Golgi
mRNA
Ribosome
Leader sequence
Membrane Ig Secreted Ig
H and L chains are synthesized on separated
ribosomes
CHAPERONES
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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
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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
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Immature B cells with specificity for multivalent self antigens are retained in the bone marrow.
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Receptor Editing of Immature B cells with self-reactive BCR (Bone Marrow)
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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)
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How can mature B-cells express surface IgM and IgD
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Co-Expression of cell surface IgM and IgGOn Mature B-cells is controlled by alternative
RNA processing
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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
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B – CELL ACTIVATION
Where and how do all these things take place?
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B-cell recycling in the absence of antigen (lymph node)
B cells in blood
Efferenslymph
T cell area
B cell area
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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
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Germinal Center Reaction
IV_10_5_Germinal_Centers-H264.mov
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„Dating” in the peripheral lymphoid organs
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The structure of the germinal centre
Somatic hypermutation
FDC
Somatic hypermutation
DZ
LZ
LZ: light zoneDZ: dark zoneFDC: follicular dendritic cell
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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
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T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID ORGANS
IgM
IgGIgAIgE