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B CELL DEVELOPMENT IN THE BONE
MARROW
ORDERED B-CELL DEVELOPMENT
immature B cell
pre B cell
ANTIGEN RECOGNIZING
RECEPTOR
H-chain + surrogate L-chain
pro B cell
H2L2
SIGNALING RECEPTOR
NO ANTIGEN RECOGNIZING
RECEPTOR
B
B
Stromal cell
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
Cell-cell contact
B
Secreted
Factors - CYTOKINES
Stromal cell
Types of cytokines and cell-cell contacts needed at
each stage of differentiation are different
Cytokines and cell-cell contacts at each stage of
differentiation are different
D-J rearranged
VLA-4
Early
pro-B
Stem
(Integrin)
Receptor
Tyrosine
kinase
Stem cell
factor
VCAM-1
(Ig superfamily)
Cell adhesion
molecules
c-Kit
Cell-bound
growth
factor
Stromal cell
No Ig
DJ rearrangement (H chain)
L-chain genes in germline form
Cytokines and cell-cell contacts at each stage of
differentiation are different
V-D-J rearranged
µ-chain made
Light chain germline
Interleukin-7
receptor
Interleukin-7
Growth factor
Early
pro-B
Late
pro-B
VLA-4
(Integrin)
VCAM-1
(Ig superfamily)
Stromal cell
Pre-B
Pre- B cell receptor
Heavy chain
VHDHJH
V-preB
CHm
l5
Iga & Igb signal
transduction
molecules
Transiently expressed when VHDHJH CHm is productively rearranged
VpreB/l5 - the surrogate light chain (SLC), is required for surface expression
Ligand for the pre-B cell receptor is unknown
Ligation of the pre-B cell receptor
1. Suppresses further H chain rearrangement
2. Triggers entry into cell cycle
Large
Pre-B
Unknown ligand of
pre-B cell receptor
1. Ensures only one specificty of
Ab expressed per cell
Stromal cell
2. Expands only the pre-B
cells with in frame VHDHJH joins
ALLELIC EXCLUSION
Ligation of the pre-B cell receptor triggers
entry into the cell cycle
100X expansion
V-J light chain
rearranged
Proliferation
Large
Pre-B
Large
Large
Pre-B
Large
Pre-B
Large
Pre-B
Pre-B
V-J light chain expression is quite
efficient with an 85% success rate
From a single µ chain 85 receptor!!!
Small
Large
pre-B
Proliferation
stops
Pre-receptor
not
displayed
Many large pre-B
cells with identical
pre-B receptors
IgM
Intracellular VDJCH chain
VL-JL rearranges
Y
Large
pre-B
Large
Pre-B
Large
Large
Pre-B
Large
Pre-B
Large
Pre-B
Pre-B
Immature
B cell
Light chain expressed
IgM displayed on surface
B cell receptor
Heavy chain
VHDHJH
Light chain
VLJLCL
CHm
Iga & Igb signal
transduction
molecules
L chain is rearranged
RECEPTOR EXPRESSION DURING B-CELL
DEVELOPMENT
Allelic exclusion
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
Evidence for allelic exclusion
ALLOTYPE- a polymorphism in the Heavy chain C region of Ig
Allotypes can be identified by staining B cell surface Ig with antibodies
B
a
B
AND b
b
B
B
Y
Suppression of H chain rearrangement by
pre-B cell receptor prevents expression of two
specificities of antibody per cell
Y
b
Y
B
Y
a
a/b
b/b
Y
Y
a/a
a
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 to prevent the emergence of
new daughter specificities during proliferation after clonal selection
Allelic exclusion prevents unwanted responses
One Ag receptor per cell
IF there were two Ag receptors per cell
Y
YY
S. aureus
Y
Y
Y
Y
Anti
S. aureus
Antibodies
B
S. aureus
Anti
Liver cell
Abs
Y
Y
Y
Y
Self antigen
expressed by
e.g. Liver cells
YY
B
Anti
S. aureus
Antibodies
Suppression of H chain gene rearrangement
ensures only one specificty of Ab expressed per cell.
Prevents induction of unwanted responses by pathogens
Allelic exclusion is needed to prevent holes in the repertoire
One specificity of Ag
receptor per cell
IF there were two specificities
of 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
B
Deletion
OR
B
BUT anti S.Aureus B cells
will be excluded
leaving a “hole in the
repertoire”
Anergy
B
S. aureus
THE RESULT OF SOMATIC GENE REARRANGEMENTS
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
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
Stages of B cell development
Stem Cell
Early pro-B cell
Late pro-B cell
Large pre-B cell
Peripheral
Y
Small pre-B cell
Immature B cell
Y
Receptor
H+L
Mature B cell
Receptor
H+L
Each stage of development is defined by IgH and IgL chain genes,
expression of adhesion molecules and cytokine receptors
SYNTHESIS OF IMMUNOGLOBULINS
Secreted Ig
Membrane Ig
Golgi
ER
H and L chains are
synthesized on separated
ribosomes
CHAPERONES
Leader sequence
Ribosome
mRNA
DEVELOPMENT OF B-LYMPHOCYTES IN THE BONE
MARROW
Limphoid precursor
B cells recognizing self
structures
Cell surface molecules
MHC proteins
Common molecules of haemopoetic
cells
c-kit/CD44
RAG-1/RAG-2
H
H rearrrangement
átrendeződés
m
apoptosis, clonal deletion
Soluble molecules
House keeping genes
Metabolites
Surrogate L
L rearrangement
B
functional unresponsiveness
anergia
Other specificites
Selection
clonal deletion
B
B
B
PERIPHERAL LYMPHOID TISSUES
Negative selection of immature
B-cells in the bone marrow
BONE
MARROW
1
2
3
4
5
n
Potential B-cell
repertoire
Self recognition
Clonal deletion
Self structure
PERIPHERAL
LYMPHOID
ORGANS
RNA editing
Foreign antigen independent
Available B-cell
repertoire
About 30 billion mature naive B cells leave the bone marrow per day
to circulate in blood
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 half
life 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 IgG
On Mature B-cells is controlled by alternative
RNA processing
RESULT OF SOMATIC GENE REARRANGEMENT AND
ALLELIC EXCLUSION
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
B – CELL ACTIVATION
Where and how do all these things
take place?
B-cell recycling in the absence of antigen
(lymph node)
T cell area
B cells
in blood
B cell
area
Efferens
lymph
Recirculating B cells are trapped by foreign
antigens in lymphoid organs
Antigen enters
node in afferent
lymphatic
YY
Y
B cells
proliferate
rapidly
B cells leave blood &
enter lymph node via
high endothelial venules
Y
YY
Y
Y
Y
GERMINAL CENTRE
Transient structure of
Intense proliferation
YY
Y
Germinal centre
releases B cells
that differentiate
into plasma cells
Germinal Center Reaction
„Dating” in the peripheral lymphoid organs
The structure of the germinal centre
Somatic hypermutation
LZ
FDC
DZ
Somatic hypermutation
LZ: light zone
DZ: dark zone
FDC: 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
IgG
IgA
IgE