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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
Stages of differentiation in the bone marrow are
defined by Ig gene rearrangement
B CELL STAGE
Stem cell
Early pro-B
IgH GENE
CONFIGURATION
Germline
DH to JH
Late pro-B Large pre-B
VH to DHJH
VHDHJH
Pre-B cell
receptor
expressed
Ig Light chain gene has not yet rearranged
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
Maturing B cells
Bone marrow stromal cell
B
B
Stromal cell
Cytokines and cell-cell contacts at each stage of
differentiation are different
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
Cytokines and cell-cell contacts at each stage of
differentiation are different
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, 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
Large pre-B cells need in frame VHDHJH joins to mature
Human IgG3 Heavy Chain
nucleotide sequence
Translation in frame 1
ATGAAACANCTGTGGTTCTTCCTTCTCCTGG
TGGCAGCTCCCAGATGGGTCCTGTCCCAGG
TGCACCTGCAGGAGTCGGGCCCAGGACTGG
GGAAGCCTCCAGAGCTCAAAACCCCACTTGG
TGACACAACTCACACATGCCCACGGTGCCCA
GAGCCCAAATCTTGTGACACACCTCCCCCGT
GCCCACGGTGCCCAGAGCCCAAATCTTGTG
ACACACCTCCCCCATGCCCACGGTGCCCAG
AGCCCAAATCTTGTGACACACCTCCCCCGTG
CCCNNNGTGCCCAGCACCTGAACTCTTGGG
AGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGATACCCTTATGATTTCCCGGACCC
CTGAGGTCACGTGCGTGGTGGTGGACGTGA
GCCACGAAGACCCNNNNGTCCAGTTCAAGT
GGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCTGCGGGAGGAGCAGTACA
ACAGCACGTTCCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAACGGCAA
GGAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGACAGCCCGAGGAGATGACCA
AGAACCAAGTCAGCCTGACCTGCCTGGTCAA
AGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAACACCACGCCTCCCATGCTGGACTCC
GACGGCTCCTTCTTCCTCTACAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACATCTTCTCATGCTCCGTGATGCATGAGGC
TCTGCACAACCGCTACACGCAGAAGAGCCTC
TCCCTGTCTCCGGGTAAATGA
MKXLWFFLLLVAAPRWVLSQV
HLQESGPGLGKPPELKTPLGD
TTHTCPRCPEPKSCDTPPPCP
RCPEPKSCDTPPPCPRCPEPK
SCDTPPPCXXCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDXXVQFKWYVDG
VEVHNAKTKLREEQYNSTFRV
VSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPEE
MTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYNTTPPMLD
SDGSFFLYSKLTVDKSRWQQG
NIFSCSVMHEALHNRYTQKSLS
LSPGK*
Translation in frame 2
(no protein)
Development
continues
Large
pre-B
Pre-B cell
receptor can
ligate to
stromal cell
Development
arrests
*
Translation in frame 3
ETXVVLPSPGGSSQMGPVPGA
PAGVGPRTGEASRAQNPTW*
Development
arrests
Stages of B cell development
Stem Cell
Early pro-B cell
Late pro-B cell
Large pre-B cell
Nagy pre-B sejt
Peripheral
Pre-receptor
Immature B cell
Y
Small pre-B cell
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
Ligation of the pre-B cell receptor triggers
entry into the cell cycle
Proliferation
Large
Pre-B
Large
Large
Pre-B
Large
Pre-B
Large
Pre-B
Pre-B
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
ORDER OF REARRANGEMENTS OF IMMUNOGLOBULIN
GENE SEGMENTS
D – J recombination
V – DJ recombination
VDJ – mδ transcription
Surrogate light chain
V – J recombination
VJ –  (or VJ - l) transcription
mδ translation
 or l translation
B-sejt
mIgD
mIgM
Secreted IgM
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
B cells have several chances to successfully
rearrange Ig genes
Early Pro B
DH-JH
On first
chromosome
NO
DH-JH
On second
chromosome
Late Pro B
YES
YES
VH-DJH
On first
chromosome
NO
VH-DJH
On second
chromosome
Pre B
YES
 on first
YES
chromosome
Y
B
YES
l on first
chromosome
IgMl
 on second
chromosome
NO
NO
NO
NO
B
IgM
YES
NO
YES
Immature B
l on second
chromosome
NO
YES
Y
B
RECEPTOR EXPRESSION DURING B-CELL
DEVELOPMENT
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
RESULT OF SOMATIC GENE REARRANGEMENT AND
ALLELIC EXCLUSION
1. Somatic rearrangement of Ig gene segments 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 huge 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
MECHANISMS INDEPENDENT ON THE PRESENCE OF
ANTIGEN
OCCUR IN THE BONE MARROW DURING B-CELL
DEVELOPMENT
SYNTHESIS OF IMMUNOGLOBULINS
Secreted Ig
Membrane Ig
Golgi
ER
H and L chains are
synthesized on separated
ribosomes
CHAPERONES
Leader sequence
Ribosome
mRNA
HOW CAN IMMATURE B CELLS EXPRESS SURFACE
IgM AND IgD IMMUNOGLOBULINS
Splicing of IgM and IgD RNA
VD J
VD J
Cm1
Cm2
Cm
Cm3
Cd
Cg3
Cd1
Cm4
Cg1
Cd2
DNA
Cd3
pA2
pA1
Two types of mRNA can be made simultaneously in the cell by differential usage of
alternative polyadenylation sites and splicing of the RNA
VD J
Cm1
Cm2
Cm3
Cm4
AAA Cd1
V D J Cm
Cd2
Cd3
RNA cleaved and
polyadenylated at pA1
IgM mRNA
RNA cleaved and
polyadenylated at pA2
VD J
Cm1
Cm2
Cm3
Cm4
Cd1
Cd2
Cd3
pA1
V D J Cd
IgD mRNA
AAA
Co-expression of cell surface IgM and IgG
on mature B-cells is controlled by alternative
RNA processing
SYNTHESIS OF IMMUNOGLOBULINS
Secreted Ig
Membrane Ig
Golgi
ER
H and L chains are
synthesized on separated
ribosomes
CHAPERONES
Leader sequence
Ribosome
mRNA