B.

Cell Development
By
Selda S. Ezzel-deen
Student PhD . Microbiology/Immunology
Generation of B Cells

• Millions of B lymphocytes are generated in the adult bone marrow every

day and exported to the periphery. The rapid generation of new B cells
occurs in a carefully regulated sequence of events. Cell transfer
experiments that identified hematopoietic stem cells (HSCs), in which
genetically marked donor HSCs are injected into unmarked recipients,
have indicated that B-cell development from HSC to mature B cell that
takes from 1 to 2 weeks.
B-cell development begins in the bone marrow with the
asymmetric division of an HSC and continues through a
series of progressively more differentiated progenitor
stages to the production of common lymphoid progenitors
(CLPs), which can give rise to B cells, T cells, or innate
lymphoid cells . The majority of CLPs that remain in the
bone marrow enter the B-cell development pathway.
 B cell development
• As differentiation proceeds, developing B cells express a precisely controlled
sequence of cell-surface receptors and adhesion molecules. Some of the signals
received from these receptors induce the differentiation of the developing B cell;
others trigger its proliferation at particular stages of development; and yet others
direct its movements within the bone marrow environment. These signals
collectively allow differentiation of the CLP through the early B-cell stages to
form the immature B cell that leaves the marrow to complete its differentiation
in the spleen.
 Stages of B cell development

Stem Cell Early pro-B cell Late pro-B cell Large pre-B cell

Peripheral

Small pre-B cell Immature B cell Mature B cell

Each stage of development is defined by rearrangements of Ig H chain genes, Ig L chain

genes, expression of surface Ig, expression of adhesion molecules and cytokine receptors
B cell development
Stages of Lymphocyte Maturation
• B-cell development begins with a hematopoietic stem cell (HSC) and passes
through progressively more committed lymphoid progenitor cell stages until
it reaches the pro-B-cell stage. At this stage, the precursor cell is irreversibly
committed to the B cell lineage, and the recombination of the
immunoglobulin genes begins. Once the completed IgM is expressed on the
cell surface, the immature B cell leaves the bone marrow to complete its
maturation through the transitional T1 and T2 stages in the spleen.
• The primary function of mature B cells is to detect pathogens and other
potentially harmful foreign antigens and to differentiate into plasma cells
secreting antibodies that protect the host against the invaders. Therefore,
the most critically important events occurring during B-cell development
are the rearrangements of immunoglobulin receptor heavy- and light-
chain gene segments to form the B-cell receptor for antigen, determining
its specificity
• B-cell development from its earliest stages in the primary lymphoid organs
to the generation of fully mature B cells in the secondary lymphoid tissues.
Most will focus on the predominant (or conventional) B-cell population

• however, several B-cell subsets exist, and differentiation of the subsets,

that is, B-1 B cells and marginal zone (MZ) B cells, differ from the
developmental program followed by the conventional B-2 B cells. We will
conclude with a brief comparison of the maturational processes of B
lymphocytes.
• That immunoglobulin gene rearrangements begin with heavy-chain D-to-J
gene segment H

• Rearrangement, followed by the stitching together of the V and DJ segments

to allow synthesis of an intact μ heavy chain. This chain initially pairs with a
surrogate light chain, allowing the cell surface expression of the pre-B-cell
receptor. This initial form of membrane immunoglobulin (Ig) activates several
rounds of cell division, followed by rearrangement of the light-chain V and J
gene segments, allowing the immature B cell to express membrane IgM.
 Regulation of B Cell Development
• Progenitor cells receive signals from bone marrow stromal cells via cell-cell contacts and

secreted signals. This bone marrow microenvironment is responsible for B cell

development. involved in both B and T cell development is SCF (stem cell factor) on the

stromal cell membrane on the lymphocyte membrane. A secreted cytokine important for

both B and T cell development is IL-7, secreted by the stromal cell and bound to IL-7R on

the developing lymphocyte. Signals from these binding events initiate cytoplasmic cascades

resulting in altered expression of proteins required for development. As the B cells develop

in the marrow, they migrate from the outer part of the marrow towards the core
B Cell Development on Stromal cell bone
marrow
 B Cell Development
Maturation - Stem cells to mature, naïve B cells

Activation- Ag binding; initiation of cell changes

Differentiation - Cell division and changes into

effectors B cells (plasma cells) and memory B cells
 Lymphopoiesis
• Occurs in yolk sac, fetal liver then bone marrow throughout rest
of life
• Mature, naïve B cells released into circulation
• B cell production occurs throughout life; does not wane as does T
cell production
• About 5 million produced per day
• Only 10% of B cells mature
• Naïve B cells survive about one week
• Undergo negative selection
Generation of Diversity
B cell receptor
(Immunoglobulin)
Heavy Light

V gene segments 100 30

D gene segments 32 -
J gene segments 6 5
N region insertion ++ -
Junctional diversity +++ +
Somatic mutation + +
VxDxJ VxJ
100 x 32 x 6 30 x 5
Total 1.9 x 104 1.5 x 102
Combinatorial association 2.9 x 106
• Ag independent process • Clonal selection
 Immunoglobulin consists of 4 polypeptide
chains
N terminus
Ag binding site Variable regions Ag binding site

VH
VL
CH1
CL

Disulfide
CH2 bonds
Constant
EM Rabbit Ig
region
X 2,042,500
CH3
Light chain

C terminus Heavy chain

 Heavy Chain Gene Family
Germ line gene organization

Heavy chain genes; Vn=100, Dn=32

L V1 L V2 L Vn D1 D2 D3 Dn J1 J2 J3 J4 J5 J6

P P P E

Cµ Cδ Cγ3 C γ1 C γ2 C γ4 Cε Cα1 Cα2

CH1 H CH2 CH3 CH4

Introns separate exons coding for H chain domains

 Heavy Chain Gene Family
Gene rearrangement and expression
L V1 L V2 L Vn D1 D2 D3 Dn J1 J2 J3 J4 J5 Cµ Cδ

P P P E

DJ rearrangement

L V1 L V2 L Vn D1 D2 J4 J5 Cµ Cδ
DNA
P P P E

VDJ rearrangement

V2 D2 J4 Cµ Cδ
L V1 L J5

P P E
DNA

Transcription
V2 D2 J4 Cµ Cδ
L J5
Primary transcript RNA
E
Light Chain Gene Families
Germ line gene organization

Lambda light chain genes; n=30

L Vλ1 L Vλ2 L Vλn Jλ1 Cλ1 Jλ2 Cλ2 Jλ3 Cλ3 Jλ4 Cλ4

P P P E E E E

Kappa light chain genes; n=35

L Vκ1 L Vκ2 L Vκn J κ1 Jκ 2 Jκ 3 J κ4 J κ5 Cκ

P P P E
Order of Ig Gene Expression
 B-Cell Development in the Bone Marrow
• During embryonic development hematopoiesis and B-cell formation occur in several
structures, including the fetal liver and spleen, beginning around the time of birth and
continuing through adulthood these key developmental pathways occur in the bone
marrow. The structure of bone and bone marrow

• The bone marrow contains microenvironments, and populated by hematopoietic cells

and various bone marrow stromal cells.

• The stromal cells express proteins (including cell-surface ligands, cytokines, and
chemokines) that support the long-term survival and division of HSCs and the
developmental pathways leading to the formation of mature blood cells.
 B-Cell Development In Bone Marrow
• HSCs and early progenitor cells express the receptor c-kit (CD117), which binds the
ligand stem cell factor (SCF, expressed both as a membrane and secreted protein),
maintaining the cells in this niche and influencing their differentiation into
progenitor cells. Once it differentiates to the pre-pro-B-cell stage, a developing B cell
requires signals from the chemokine CXCL12, secreted by certain stromal cells, in
order to progress to the pro-B-cell stage. Pro-B cells then require signaling from the
cytokine interleukin (IL)-7, which is secreted by yet another stromal cell subset, to
mature to the pre-B stage . Many of these stromal cell factors serve to induce the
expression of specialized transcription factors important in B-cell development.
 The Cell-Surface Markers, Gene Expression, and Immunoglobulin
Gene Rearrangements in Stages of B-Cell Development

• The developmental pathway requires that scientists understand the phenotypic

and functional characteristics of each cell type in that pathway

• Cells at particular stages of differentiation can be characterized by their surface

molecules, which include adhesion molecules and receptors for chemokines
and cytokines.

• They are also defined by the array of active transcription factors that determine
which genes are expressed at each step in the developmental B Cell process
Role of Genetic and Epigenetic IN B.Cell
Development
• In the immune system, like any developing system, the control of expression of certain
genes and their protein products is of key importance to the stepwise differentiation of
stem and progenitor cells into mature differentiated cells. How each step in the
progression of cells from early hematopoiesis through B-cell development is controlled is
a major question occupying immunologists and is of interest to clinicians as well, as
defects in this process can lead to immunodeficiencies or malignancies (leukemias and
lymphomas).

• Several transcription factors have been identified in the that control of gene expression
during B-cell development and the regulatory mechanisms control by gene transcription
and translation.
 Roles of Epigenetic Changes in the Control
of B-Cell Development
• the control of B Cell development has also uncovered critical roles played by
epigenetic changes (changes affecting a gene’s expression that don’t affect the
DNA sequence of the gene itself). These findings have emerged from molecular
analyses of the DNA and chromatin associated with specific genes and their
modifiers. Epigenetic changes include DNA modification by methylation,
chromatin alterations such as histone modification and chromatin structure.

• remodeling, and the effects of microRNAs, which can inhibit protein expression by
decreasing the stability of mRNAs and their translation into protein.
• Bone marrow stromal cell–derived proteins, including stem cell
factor (SCF) and IL-7, induce early hematopoietic cells to become
increasingly committed to the lymphoid lineage.

• A network of sequentially activated transcription regulators

drives hematopoietic differentiation, generating common
lymphoid progenitors that give rise to the B-lymphocyte lineage.
The Later Stages of B-Cell Development to B-Cell Phenotype
and the Stepwise Rearrangement of Immunoglobulin Genes

• including expression of key proteins and immunoglobulin heavy- and light-

chain gene rearrangements—for stages of B-cell development beginning with
the first cell committed to the B-cell lineage

• The first, and most widely used, is the Basel nomenclature (pre pro-B, early
and late pro-B, large and small pre-B, immature B).

• The second (A, B, C, C′, D, E) is that the experiments that defined this system
of classification of B-cell development .
The Stages of B-Cell Development(Hardy
Fractions)
• Pre-Pro-B Cells
• the B-cell lineage–specific marker B220 (CD45R), and the expression of
increasing levels of the transcription factor EBF1, the developing
common lymphoid progenitor enters the pre-pro-B-cell stage. EBF1 is an
important transcription factor in lymphoid development, and therefore
transcription of the Ebf1 gene is itself under the control of multiple
transcription factors.
• Stages of B-cell development can also be defined by the status of
immunoglobulin gene rearrangements. The heavy-chain V genes
rearrange first in pre-pro- and pro-B cells, with D-to-J recombination
occurring initially, followed by V -to-DJ recombination.

• The heavy chain is then expressed on the cell surface in combination with
the surrogate light chain, which is made up of V pre B and λ5. Together
they form the pre-B-cell receptor, which is expressed on the cell surface
along with the Igα/Igβ signaling complex.
Completion of B-Cell Development in the
Spleen
• Immature B cells are recruited to leave the bone marrow by their
expression of the S1P receptor, which recognizes the lipid
chemoattractant sphingosine 1-phosphate (S1P) in the blood

• These cells then migrate to the spleen, where they complete their
development into mature B cells.
 B Cell Subsets
• B1
• CD5 marker
• More responsive to CHO Ags
• Seen in peritoneum
• B2
• Most B cells (95%)
• Drive the Ag response in secondary lymphoid organs
 T1 and T2 Transitional B Cells Form in the
Spleen To Survival and against Self-Reactivity
• T1 and T2 transitional B cells in the spleen were initially characterized on the basis of their
cell surface expression of immunoglobulin receptors and other membrane markers .

• T2 B cells differ from T1 B cells in having higher levels of membrane IgD and in expressing
CD21 (the complement receptor and B-cell coreceptor; and CD23.

• T2 B cells also express the BAFF-receptor (BAFF-R), a receptor for B-cell survival factor
BAFF (B-cell activating factor belonging to the tumor necrosis factor family); BAFF-R
expression is dependent on signals received through the BCR.
T2 B Cells Give Rise to Mature Follicular B-
2 B Cells
• Fully mature conventional B-2 cells express high levels of IgD and
intermediate levels of IgM on their cell surface.

• T2 B cells mature into conventional B-2 cells that populate the follicles
of lymph nodes and spleen, and hence are called follicular B cells.
T3 B Cells Are Primarily Self-Reactive and
Anergic
• T3 transitional B cells were first characterized in the blood and
lymphoid organs by flow cytometry, and were described as being
CD93 mIgD mIgM CD23 .

• Recent experiments have suggested that the T3 population may

represent transitional B cells that have been rendered anergic by
contact with soluble self antigen in the spleen.
 Conclusion
• The first essential challenge that must be accomplished in B-cell development is the
generation of B cells with a vast repertoire—many billions—of B-cell receptor
specificities that are sufficient to ensure responses to virtually anything foreign that
enters the body. The antibody diversity generated by gene rearrangements, junctional
diversification, and different combinations of heavy and light chains.

• the amplified by the fact that millions of new B cells are generated every day. B cells
whose antibody specificities aren’t needed turn over, replaced by new B cells generated
in the bone marrow by the processes of hematopoiesis and B-cell development.