1. B CELL DEVELOPMENT IN THE BONE MARROW

2. ORDERED B-CELL DEVELOPMENT immature B cell pre B cell pro B cell
ANTIGEN RECOGNIZING RECEPTOR
H2L2
pro B cell
H-chain + surrogate L-chain
SIGNALING RECEPTOR
NO ANTIGEN RECOGNIZING RECEPTOR

3. B B
Stromal cell

4. Bone marrow stromal cells nurture developing B cells
1. Specific cell-cell contacts between stromal cells and developing B cells
Cell-cell contact
Secreted
Factors - CYTOKINES
2. Secretion of cytokines by stromal cells B
Stromal cell
Types of cytokines and cell-cell contacts needed at each stage of differentiation are different

5. Cytokines and cell-cell contacts at each stage of differentiation are different
D-J rearranged
Stem
Early pro-B
c-Kit
Receptor Tyrosine
kinase
Stem cell factor
Cell-bound growth
factor
VLA-4
(Integrin)
Cell adhesion
molecules
VCAM-1
(Ig superfamily)
Stromal cell
No Ig
DJ rearrangement (H chain)
L-chain genes in germline form

6. Cytokines and cell-cell contacts at each stage of differentiation are different
V-D-J rearranged
Light chain germline
µ-chain made
Interleukin-7
receptor
Interleukin-7
Growth factor
Early pro-B
VLA-4
(Integrin)
VCAM-1
(Ig superfamily)
Late pro-B
Pre-B
Stromal cell

7. Ligand for the pre-B cell receptor is unknown
CHm
Heavy chain
VHDHJH
V-preB 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

8. 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

9. Ligation of the pre-B cell receptor triggers entry into the cell cycle
Large
pre-B
100X expansion
Large
Pre-B
Many large pre-B cells with identical pre-B receptors
Proliferation
V-J light chain
rearranged
V-J light chain expression is quite efficient with an 85% success rate
From a single µ chain 85 receptor!!!
Large pre-B Y
Immature
B cell
Light chain expressed
IgM displayed on surface
IgM
Proliferation stops
Pre-receptor not displayed
Small pre-B
Intracellular VDJCH chain
VL-JL rearranges

10. B cell receptor Heavy chain VHDHJH Light chain VLJLCL CHm
Iga & Igb signal
transduction
molecules
L chain is rearranged

11. RECEPTOR EXPRESSION DURING B-CELL DEVELOPMENT

12. 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

13. 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
a/a
b/b
a/b Y B a Y B b Y B a Y B b
AND Y B a b
Suppression of H chain rearrangement by pre-B cell receptor prevents expression of two specificities of antibody per cell

14. 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

15. Allelic exclusion prevents unwanted responses
One Ag receptor per cell
IF there were two Ag receptors per cell Y B Y
Self antigen
expressed by
e.g. Liver cells B Y
S. aureus
S. aureus Y
Anti
S. aureus
Antibodies Y
Anti
Liver cell
Abs Y
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

16. Allelic exclusion is needed to prevent holes in the repertoireY B
One specificity of Ag receptor per cell Y B
IF there were two specificities of Ag receptor per cell
Anti-brain Ig
Anti-brain Ig
AND
anti-S. Aureus Ig
Exclusion of anti-brain B cells i.e. self tolerance
BUT
anti S.Aureus B cells will be excluded leaving a “hole in the repertoire” B
Deletion
Anergy OR Y B
S. aureus

17. THE RESULT OF SOMATIC GENE REARRANGEMENTS
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

18. 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

19. 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
Mature B cell
Receptor
H+L Y
Receptor
H+L
Each stage of development is defined by IgH and IgL chain genes, expression of adhesion molecules and cytokine receptors

20. SYNTHESIS OF IMMUNOGLOBULINS
Secreted Ig
Membrane Ig
Golgi ER
H and L chains are synthesized on separated ribosomes
CHAPERONES
Leader sequence
Ribosome
mRNA

21. 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
apoptosis, clonal deletion
Soluble molecules
House keeping genes
Metabolites
functional unresponsiveness
anergia
c-kit/CD44
RAG-1/RAG-2
H rearrrangement
H átrendeződés 
Surrogate L
L rearrangement B
Selection
clonal deletion B
Other specificites B B
PERIPHERAL LYMPHOID TISSUES

22. Negative selection of immature B-cells in the bone marrow
Potential B-cell repertoire
BONE MARROW
Self structure
Self recognition
Clonal deletion
RNA editing
PERIPHERAL LYMPHOID ORGANS
Available B-cell repertoire
Foreign antigen independent
Hozzáférhető sokféleség….
About 30 billion mature naive B cells leave the bone marrow per day
to circulate in blood

23. Immature B cells with specificity for multivalent self antigens are retained in the bone marrow.
Immature B cells that are not specific for a self antigen in bone marrow mature further to express IgM and IgD and leave the bone marrow (left panels). Immature B cells specific for a self antigen on bone marrow cells are retained in the bone marrow (right panels).

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

25. 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)

26. How can mature B-cells express
surface IgM and IgD

27. Co-Expression of cell surface IgM and IgG
On Mature B-cells is controlled by alternative
RNA processing

28. RESULT OF SOMATIC GENE REARRANGEMENT AND ALLELIC EXCLUSION
Somatic rearrangement of Ig gene segments occurs in a highly controlled manner
Single B-cells become committed to the synthesis of one unique H-chain and one unique L-chain variable domain, which determine their specificities
In one individual a large B-cell repertoire is generated consisting of B-cell clones with different H- and L-chain variable domains
This potential B-cell repertoire is able to recognize a wide array of various antigens
Immature B-cells express IgM and IgD surface Ig with the same variable domains

29. Where and how do all these things take place?
B – CELL ACTIVATION
Where and how do all these things take place?

31. B-cell recycling in the absence of antigen
(lymph node)
B cells
in blood
T cell area
B cell area
Efferens
lymph

32. Recirculating B cells are trapped by foreign antigens in lymphoid organs
B cells leave blood & enter lymph node via
high endothelial venules
B cells
proliferate
rapidly
Antigen enters
node in afferent
lymphatic Y
Germinal centre
releases B cells
that differentiate
into plasma cells
GERMINAL CENTRE
Transient structure of
Intense proliferation

33. Germinal Center Reaction
Germinal centers, where B cells proliferate and undergo both isotype switching and somatic hypermutation, form within the B cell follicles in lymphoid organs. The formation of germinal centers starts when dendritic cells displaying antigen on their surface activate antigen-specific CD4 T cells, which proliferate and mature into effector cells capable of activating antigen-specific B cells. Once activated by a CD4 T cell, the B cell proliferates to form a primary focus of antigen specific B cells. B cells from the primary focus migrate to nearby follicles and proliferate. Other B cells from the primary focus persist in the T cell area for a short while, secreting antibody, but eventually die. The B cells that enter the follicle begin to proliferate rapidly. During this time they also undergo somatic mutation, to introduce new variation into the B cell receptor. B cells undergo a process of selection after somatic mutation, when the receptors are tested for their ability to bind antigen. Those that fail to bind, or that fail to compete efficiently against other B cell receptors, die.

34. „Dating” in the peripheral lymphoid organs

35. The structure of the germinal centre
Somatic hypermutation LZ
FDC DZ
Somatic hypermutation
LZ: light zone
DZ: dark zone
FDC: follicular dendritic cell

36. Antigen is bound on the surface of follicular dendritic cells (FDC)
 FDC-s bind immune complexes (Ag-Ab )
 Ag detectable for 12 months following immunization
 A single cell binds various antigens
Fig 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
B cells recognize Ag on the surface of FDC
This concept is being challenged now, and there has been some backtracking in the current text. It is possible that the FDCs provide persistent Ag to long-lived plasma cells.

37. T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID ORGANS
IgM
IgG
IgA
IgE