1. ANTIGEN-INDEPENDENT DEVELOPMENT
OF B-LYMPHOCYTES

2. The development of B cells can be divided into six
functionally distinct phases
The first two phases correspond to development in the bone marrow; the last four phases correspond to development in the secondary lymphoid tissues.
bone marrow
secondary lymphoid tissues

3. B cells develop in bone marrow and then migrate to
secondary lymphoid tissues
B cells leaving the bone marrow (yellow) are carried in the blood to lymph nodes, the spleen, Peyer’s patches (all shown in red), and other secondary lymphoid tissues such as those lining the respiratory tract (not shown).

4. Pro-B cells develop from the pluripotent hematopoietic
stem cell
Cells at different stages of development are identified by different combinations of CD proteins on their surface. CD127 is the α chain of the receptor for interleukin-7.
IL7-Rα

5. The early stages of B-cell development are dependent on bone marrow stromal cells
60 billion B cells/day
The early stages of B-cell development are dependent on bone marrow stromal cells.
The panels show the interactions of developing B cells with bone marrow stromal cells. Stem cells and early pro-B cells use the integrin VLA-4 to bind to the adhesion molecule VCAM-1 on stromal cells. This and interactions between other cell-adhesion molecules (CAMs) promote the binding of the receptor Kit on the B cell to stem-cell factor (SCF) on the stromal cell. Activation of Kit causes the B cell to proliferate. B cells at a later stage of maturation require interleukin-7 (IL-7) to stimulate their growth and proliferation.
Middle panel: Panel a is a light micrograph of a tissue culture showing small round B-cell progenitors in intimate contact with stromal cells, which have extended processes fastening them to the plastic dish on which they are grown. Panel b is a high-magnification electron micrograph showing two lymphoid cells (L) adhering to a flattened stromal cell. Photographs courtesy of A. Rolink (a); Paul Kincade and P.L. Witte (b).
The development of B cells in the bone marrow proceeds through stages defined by the rearrangement and expression of the immunoglobulin genes
In the stem cell, the immunoglobulin (Ig) genes are in the germline configuration. The first rearrangements are of the heavy-chain (H-chain) genes. Joining DH to JH defines the early pro-B cell, which becomes a late pro-B cell on joining VH to DJH. Expression of a functional μ chain defines the large pre-B cell. Large pre-B cells proliferate, producing small pre-B cells in which rearrangement of the light-chain (L-chain) gene occurs. Successful light-chain gene rearrangement and expression of IgM on the cell surface define the immature B cell.

6. Various cell adhesion molecules cytokines and transcription factors regulate B cell development
PU.1
Ikaros
EBF, E2A
Pax5
CLP
early
pro-B
c-Kit
Receptor
Stem cell factor (SCF)
Cell membrane bound
VLA-4
(Integrin)
adhesion
molecules
VCAM-1
(Ig superfamily)
Stroma cell
Cell-fate specification of multipotent haematopoietic stem cells (HSCs) is determined by unique expression patterns of combinations of transcription factors, and for B cells, the five most important factors are PU.1, Ikaros, E2A, EBF (early B-cell factor) and PAX5 (paired box protein 5).

7. Cytokines and cell adhesion molecules change with successive steps of development
Interleukin-7
receptor
Interleukin-7
Growth factor
Early
pro-B
VLA-4
(Integrin)
VCAM-1
(Ig superfamily)
Late pro-B
Pre-B
Interleukin-7 (IL-7) is a key cytokine during B cell development and is produced by stromal cells in the bone marrow. IL-7 receptor (IL-7R) signaling leads to the proliferation and survival of B cell progenitors as well as aids in the commitment of cells to the B lineage. Mice with targeted deletions of IL-7 or the IL-7R display a severe block at the early pro-B cell stage of development.
Stromal cell

8. Immunoglobulin heavy-chain gene rearrangement in
pro-B cells gives rise to productive and nonproductive
rearrangements
A productive rearrangement enables the B cell to proceed to the next stage of development. Rearrangements occur at the H-chain genes on both chromosomes, and if neither is successful the cell dies.

9. The pre B-cell receptor monitors the quality of heavy chain rearrangement
FIRST checkpoint!
Mutation in λ5– arrest at Pro-B cell
stage
SEVERE IMMUNODEFICIENCY
Productive µ-chain rearrangement
- Assembly of pre-BCR
- Switch off RAG genes, enzymes
No further µ-chain rearrangement
ALLELIC EXCLUSION
Only one specificity
Distinguishing the pre-B-cell receptor from the B-cell receptor is the absence of a κ or λ immunoglobulin light chain, and the presence instead of the surrogate light chain composed of the VpreB and λ5 polypeptides. In low abundance at the cell surface, the pre-B-cell receptor is largely retained inside the cell in membrane-enclosed vesicles, from where it generates signals that lead to the cessation of heavy-chain gene rearrangements. In addition to forming the two Ig-like domains of the surrogate light chain, VpreB and λ5 have extensions that cause oligomerization of pre-B-cell receptors and the transduction of signals necessary for pre-B-cell survival.

10. Successful somatic rearrangement in one chromosome
. ALLELIC EXCLUSION
Successful somatic rearrangement in one chromosome
inhibits gene rearrangement in the other chromosome

11. Allelic exclusion at the immunoglobulin loci gives rise
to B cells having antigen receptors of a single specificity
The top panel shows the binding to antigen of B-cell receptors produced in a B cell expressing immunoglobulin from one immunoglobulin heavy-chain locus and one immunoglobulin light-chain locus only. All the receptors have identical antigen-binding sites and bind their antigen with high avidity. The bottom panel shows the B-cell receptors formed in a hypothetical B cell expressing immunoglobulin from both the immunoglobulin heavy-chain loci and one light-chain locus. Hybrid immunoglobulins are formed with disparate antigen-binding sites and bind the antigen poorly and with low avidity. The disparities would be even greater in a B cell expressing two heavy-chain and two light-chain genes.

12. A genetic model provides 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

13. 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

14. Allelic exclusion prevents unwanted responses
One Ag receptor per cell
IF there were two Ag receptors per cell Y B Y
Self antigen B Y
S. aureus
S. aureus Y
Anti
S. aureus
Antibodies Y
Anti
self
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

15. 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-self 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 B OR Y B
Deletion
Anergy
S. aureus

16. Y Y Assembly of the pre-B cell receptor induces cell proliferation
Large
Pre-B
Large
Pre-B
About 100 large pre-B cells
Large
Pre-B
Large
Pre-B
Large
pre-B
Proliferation
Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B
RAG off
RAG on Y
Immature
B cell
L chain expressed
Membrane-bound IgM
IgM
Large pre-B
smallpre-B
Intracellular VDJCH chain
VL-JL rearrangement
proliferation stops
Pre-B receptor disappears

17. Nonproductive light-chain gene rearrangements can be
superseded by further gene rearrangement
The organization of the light-chain loci allows initial nonproductive rearrangements at one locus to be followed by further rearrangements of that same locus that can lead to the production of a functional light chain. This type of rescue is shown for the κ light-chain gene. After a nonproductive rearrangement of Vκ to a Jκ, in which the translational reading frame has been lost, a second rearrangement can be made by Vκ2, or any other Vκ that is on the 5′ side of the first joint, with a Jκ that is on the 3′ side of the first joint. When the second joint is made, the intervening DNA containing the first joint is excised. There are five Jκ gene segments and many more Vκ gene segments, so that as many as five successive attempts at productive rearrangement can be made at each of the two κ light-chain loci.

18. Rearrangement of the immunoglobulin light-chain genes
in pre-B cells leads to the expression of cell-surface IgM
If a nonproductive rearrangement is made on one chromosome of a homologous pair, then rearrangement is attempted on the second chromosome.

19. COMMITMENT TO ONE TYPE OF ANTIGEN BINDING SITE
One B cell produces only one type of heavy and one type
of light chains
COMMITMENT TO ONE TYPE OF ANTIGEN BINDING SITE
B cells that are not able to express functional BCR
die by apoptosis

20. B-cell development in the bone marrow
There are two fate-determining checkpoints during
B-cell development in the bone marrow
Both checkpoints are marked by whether a functional receptor is made, which is a test of whether a functional heavy chain (at the first checkpoint) or a functional light chain (at the second checkpoint) has been produced. Cells that fail either of these checkpoints die by apoptosis.

21. Comparison of the properties of B-1 cells and B-2 cells
B-1 cells develop in the omentum, a part of the peritoneum, as well as in the liver in the fetus, and are produced by the bone marrow for only a short period around the time of birth. A pool of self-renewing B-1 cells is then established, which does not require the microenvironment of the bone marrow for its survival. The limited diversity of the antibodies made by B-1 cells and their tendency to be polyspecific and of low affinity suggests that B-1 cells produce a simpler, less adaptive immune response than that involving B-2 cells.

22. B lymphocyte subsets
A, Most B cells that develop from fetal liver–derived stem cells differentiate into the B-1 lineage. B, B lymphocytes that arise from bone marrow precursors after birth give rise to the B-2 lineage. Two major subsets of B lymphocytes are derived from B-2 B cell precursors. Follicular B cells are recirculating lymphocytes; marginal zone B cells are abundant in the spleen in rodents but can also be found in lymph nodes in humans.

23. Immature B cells specific for soluble, monovalent
NEGATIVE SELECTION I.
Immature B cells specific for soluble, 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)
Anergy: A state of unresponsiveness to antigenic stimulation. Lymphocyte anergy (also called clonal anergy) is the failure of clones of T or B cells to react to antigen
and is a mechanism of maintaining immunologic tolerance to self. Clinically, anergy describes the lack of T cell–dependent cutaneous delayed-type hypersensitivity
reactions to common antigens.

24. antigens are retained in the bone marrow
NEGATIVE SELECTION II
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).

25. By changing their antigen specificities receptor editing
rescues many self-reactive B cells
Binding to a self antigen in the bone marrow causes the immature B cell to continue to rearrange its light-chain loci. This deletes the existing self-reactive rearrangement and may also produce a new light-chain rearrangement giving immunoglobulin that is not specific for self antigen. Successive rearrangements occur until either a new non-self-reactive rearrangement is made, and the B-cell continues its development (right panels), or no more rearrangements are possible and the B cell dies (left panels).

26. The B cell pool consists of B cells with differently
rearranged immunoglobulin genes

27. Co-Expression of cell surface IgM and IgD
on mature B-cells is controlled by alternative
RNA processing
Alternative processing of a primary RNA transcript results in the formation of a μ or δ mRNA. Dashed lines indicate the H chain segments that are joined by RNA
splicing.

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

29. B-cell circulation through a lymphoid tissue
B cells circulating in the blood enter the lymph node cortex via a high endothelial venule (HEV). From there they pass into a primary lymphoid follicle. If they do not encounter their specific antigen, they leave the follicle and exit from the lymph node in the efferent lymph. The circulation route is the same for immature and mature B cells, which all compete with each other to enter primary follicles.

30. Immature B cells must pass through a primary follicle
POSITIVE SELECTION
Immature B cells must pass through a primary follicle
in a secondary lymphoid tissue to become mature B cells
Immature B cells enter the secondary lymphoid tissue through the walls of HEVs, attracted by the chemokines CCL21 and CCL19, and compete with other immature and mature B cells to enter a primary follicle (PF). Follicular dendritic cells (FDCs, turquoise) secrete the chemokine CXCL13, which attracts B cells into the follicle. Immature B cells that enter a follicle interact with proteins on FDCs that signal their final maturation into mature B cells. If they do not encounter their specific antigen in the follicle, mature B cells leave the lymph node and continue recirculating through the secondary lymphoid tissues via lymph and blood. Immature B cells that fail to enter a follicle also continue recirculation but soon die.

31. 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

32. Summary of the main stages in B-cell development
The top panels summarize the early stages of development in the bone marrow. The status of the immunoglobulin heavychain (μ) and light-chain (κ/λ) genes is shown below each panel. The bottom panels summarize the development of B cells after they leave the bone marrow, enter secondary lymphoid tissues and are activated by pathogen-derived specific antigen.