#immunology #bcell

2.  B cells( B lymphocytes), are a type of white blood cell of the
lymphocyte subtype. It comes from the name of bursa of
fabricius in bird.
 B cells are generated in the bone marrow
 Takes 1-2 weeks to develop from hematopoietic
stem cells to mature B cells

3. Hematopoietic stem cells (HSC)
Common lymphoid progenitor
(CLP)
Some of them migrate to
thymus & Tcells will be
formed
Majority of them remain
in bone marrow
&differentiated to B cells
Common myeloid
progenitor(CMP)

4. B- cell generation, activation and differentiation
Ag independent phase : A
process is an orderly
sequence of Ig-gene
rearrangements, which
progresses in the absence of
antigen in bone marrow.
Ag dependent phase
A process occur in the
periphery require Ag for
activation and differentiation .
 A mature B expressing
membrane-bound
immunoglobulin (mIgM and
mIgD) with single antigenic
specificity .
 affinity maturation is the
progressive increase in the
average affinity of the Ab
produced
 class switching is the
change in the isotype of the
Ab produced by the B cell
from µ, , or

5.  Bone-marrow stromal cells are required for maturation of progenitor
B cells(pro-B cells) into precursor B cells(pre-B cell).
 Pro-B cells bind to stromal cells by means of an interaction between
VCAM-1 on the stromal cell and VLA-4 on the pro-B cell.
 This interaction promotes the binding of c-Kit on the pro-B cell to
stem cell factor (SCF) on the stromal cell, which triggers a signal,
mediated by the tyrosine kinase activity of c-Kit
 stimulates the pro-B cell to express receptors for IL-7. IL-7 released
from the stromal cell then binds to the IL-7 receptors, inducing the
pro-B cell to mature into a pre-B cell.
 Pre-B cells completes its differentiation &leave the bon marrow as
immature B-cells.

7.  During early stages of B cell development, functional
rearrangements of the heavy chain gene locus (IgH) allows for
assembly of the pre-B cell receptor complex cessation of
IgH rearrangements (allelic exclusion) light chain
rearrangements (kappa, then lambda)
 Production of complete Ig (2 heavy chains, 2 light chains)
allows for assembly of mature B cell receptor on the cell
surface signals cessation of light chain gene rearrangement
mature B cell stage

9. •IL-7 stimulate STAT5
transcriptional factors.
•STAT5 activate the proliferating
control proteins C-myc & N-myc
that enhance B-cells proliferation..
•STATA with E2A proteins
promotes the expression of early
B cells factors 1 (EBF1).
• EBF1 in turn promotes the
expression of PAX5 , and together
the E2A proteins , EBF1,and
PAX5 activate many genes
leading to B-cell lineage
specification .

10.  Express CD45R, a B-cell lineage-specific marker.
 Increase expression of EBF-1(early B-cell factor 1).
 EBF-1 and E2A bind the Ig gene, promoting
accessibility of D-JH locus, preparing for the 1st
step in Ig gene recombination.
 EBF-1 also important for expression of other B cell
proteins, including CD79α/β and genes encoding
the pre-B cell receptor.

11.  D-JH recombination complete
 Require IL-3, IL-7, insulin-like growth factor 1, stem cell factor
 Pax5 expression (target of EBF-1)
• Master transcription factor (essential for all subsequent stages of
B cell development)
• Promotes VH to D recombination
 RAG1 and RAG2 expression
Catalyze D-JH rearrangements within the Ig heavy chain gene .
 Surface expression of CD19
• Component of multimolecular surface complex involved in
signaling in response to antigen and T cell help
 Expression of HLA-DR and CD34
 By late pro-B cell stage, most cells have initiated VH to DJH Ig
gene segment recombination.

12.  Ig heavy chain genes complete V-D-J recombination
–Allows surface expression of Ig heavy chain and surrogate light chains complex = pre-B cell
receptor
 CD79a and CD79b (Ig-α and Ig-β)
–signal transducing components of the pre-B cell receptor
–components of the Ig receptors on the surface of mature B cells
 Signaling through the pre-B cell receptor induces a few rounds of proliferation; at the end
of this the pre-B cell receptor is lost from the surface late pre B cell stage
 If pre B cell receptor cannot be displayed on cell surface because of nonproductive
VHDJH gene rearrangement, then B cell development stops and the cell undergoes
apoptosis (1st checkpoint)
 Pre-B cell receptor signaling causes transient decrease in RAG1/2 and loss of Tdt
 Ensures that as soon as one heavy chain gene has been rearranged, no further
recombination is possible (allelic exclusion)
 Light chain rearrangement is initiated following re-expression of RAG1/2
 Once light chain rearrangement has been successfully completed, the intact IgM receptor
can be expressed
 –If light chain rearrangement does not occur successfully, then the 2nd checkpoint occurs

13.  Have functional IgM but no other Ig expression
 Express B220, CD25, IL-7R, CD19.
 Once there is a functional BCR on the membrane, it has to be tested
for its ability to bind self-antigens to ensure that few auto-reactive B
cells are released.
 Three fates if autoreactive
1-Clonal deletion via BCR-mediated apoptosis
2-Reactivation of RAG to initiate process of light chain receptor editing
3-Survive and escape the BM but become anergic
 B cell loss prior to leaving the BM = central tolerance
 Export to spleen where further development occurs
 Very susceptible to tolerance induction

14. Sequence of Events and Characteristics of the
Stages
in B-cell Maturation in the Bone Marrow
Sequence of Events and Characteristics of the
Stages
in B-cell Maturation in the Bone Marrow
lymphoid pro-B pre-B immature
mature
stem cell cell cell B cell
B cell
mIgM
+
mIgD
(IL-2R a)
CD45R: a protein tyrosine phosphatase
CD19: part of the B-cell coreceptor
CD43: leukosialin
CD24: heat-stable antigen (HAS)

15. Immature B cells migrate from
bone marrow to spleen
Immature B cells classification
into two subpopulation of
transitional B cells(T1,T2)
Precursors to the fully mature B
cells.

16. T1: mIgMhi, mIgD-/lo, CD21-, CD23-,
CD24+, CD93+
T2: higher levels of mIgD, CD21+,
BAFF-R
T1 T2 mature B cells
Most T1 transitional B cells differentiate
to T2 within the spleen but ~25% of T2
emerge directly from the BM
T2 cells capable of recirculation among
the blood, lymph nodes, spleen
T2 cells can enter B cell follicles

17. Self-reactive T1 B cells eliminated by
apoptosis in response to strong
antigenic signal (peripheral tolerance)
55-75% of immature B cells lost this
way
T2 cells become resistant to antigen-
induced apoptosis Increased Bcl-X1
expression
BAFF receptor expression first
detected in T1 B cells, increases
thereafter Promotes survival of
transitional B cells

18.  Complete IgM molecule on cell surface
 Mature resting B cells express HLA-DR, CD19, CD20, CD40 but no
longer express CD10, CD34, RAG1, RAG2, or Tdt
 Exit bone marrow, migrate to secondary lymphoid organs, then
express both surface IgM and IgD as well as other molecules that
mediate cell-cell and cell-ECM adhesive interactions
 Can recirculate between blood and lymphoid organs, entering B cell
follicles in lymph nodes and spleen, responding to antigen
encounter with T cell help, leading to antibody production.
Plasma cells  class switching
Memory B cells  class switch but non-proliferating, long-lived

19. Follicular (B-2)
B cells
(conventional B
cells)
B-1 B cells
Marginal Zone
B cells

20.  30-50% of the B cells in the pleural and peritoneal cavities,
only small % of splenic B cell population
 Limited receptor repertoire, primarily directed towards
commonly expressed microbial carbohydrate antigens
 Transitional B-1 cells undergo apoptosis unless they interact
with self-antigens
◦ Constantly regenerated in the periphery Bone marrow
ablation causes depletion of B-2 pool but not B-1 pool
 Exposure to antigen leads to plasma cell formation and to
clonal expansion and persistence of antigen-specific memory
B1 cells

21.  Located in outer zones of white pulp of the spleen
 Derived from the T2 transitional population
 Specialized for recognizing blood-borne antigens
 Can respond to both protein and carbohydrate antigens
 Produce broadly cross-reactive IgM antibodies
 High levels of membrane IgM and CD21, low levels of
membrane IgD and CD23
 Long lived

23. Two major types:
A- thymus-dependent (TD) antigens
involves protein antigens and CD4+ helper T cells
 Multivalent antigen binds and cross-links membrane Ig
receptors
 Activated T cell binds B cell thru antigen receptor and via
 CD40L (T)/CD40 (B) interaction
B- thymus-independent (TI) antigens
 involves multivalent or highly polymerized antigens, does not
require T cell help
 TI-1: e.g., LPS.activate B cells that bind the antigen via the Ig
receptor
 –TI-2: e.g., bacterial capsular polysaccharide.Many are bound by
C3d

25.  BCR consist of an antigen-binding mIg and one signal-
transducing Ig-α /Ig-β heterodimer.
 The immunoreceptor tyrosine-based activation motifs (ITAMs)
are activating with several members of the Src family of
tyrosinekinases (Lyn, Blk, and Fyn).
 Assembly of large signaling lead to creating docking sites for
Syk kinase , which is activated.
 Changes in gene expression.
 The B-Cell-Co receptor Complex Can Enhance B-Cell
Responses : TAPA-1 (CD81), CR2 (CD21), and CD19 for
destroying invaders.

27.  b-cells bind Ag via Ig receptors.
 B-cells processed this Ag & re-expressed to
form peptide presented by MHC II.
 T-cells interact with B cells by CD40L on T-
cells & CD40 on B-cells,CD28 co receptors
on T-cells engaged with (B7) CD 80 or
CD86 on B-cells.
 Induced T-cells to release activating
cytokine for B cells activation.

28.  some activated B cells differentiate into plasma cells are outside of
the follicles, then migrate to the lymph node or the bone marrow.
Secrete IgM within 4 days
 Other activated B cells enter the follicle, divide and differentiate;
germinal centers form.
 •Ig genes undergo class switching: the μ constant regions replaced
by other constant regions and the variable region is subject to
somatic hypermutation.
 •Mutated variable region subject to antigen-mediated selection
 •Low affinity and autoreactive B cells die while those with improved
affinity leave the germinal centers.
 •Antibodies with mutations in the variable region appear in the
circulation within 6-10 days

29. Marginal Zone
Germinal Center
Naïve Immigrant
From Marrow
Low affinity IgM
High affinity IgG
Memory Cell
antigen
IgM
MHCII
IgG
2. FDC-
1. Ag-sp T cell
1.Affinity
Maturation
2. Isotype
Switching

30.  Introduce point mutation at very high rate
into the variable region of rearranged heavy
chian and light chain genes VDJ.
 Produced Ab with high affinity to Ag during
process of the immune response.
 Abs with mutation in their variable region
appears in the circulation 6 to 10 days
following onset of the immune response.

31. Class switching :is change a B cells production of immunoglobulin from one
type to another.
• The constant region of the Abs heavy chain is changed
• The variable region of heavy chain stays the same.
• Don’t affect Ags specificity , but the Abs retains affinity for the same Ags.
• Occurs only during active immune response
 RNA splicing
 DNA recombination

32.  To switch the IgM expression of any of the
other classes of Abs except δ heavy chain
results in irreversible loss of the intervening
of DNA.
 The formation of γ ,Є and α heavy chain
genes require cutting & re joining of the
heavy chain DNA.
 Genes for constant region lies down stream
from rearranged VDJ region.

33. RNA SPLICING
 Naïve B-cells could simultaneously expressed
both IgM & IgD.
 Both Igs are encoded on the same transcript.
 µ( IgM) versus δ (IgD) genes for heavy chain is
made by level of RNA splicing.

34. VDJ δ
VDJ µ δ
VDJ
µ
RNA
TRAS
Ig
D
IgM

36. AID
(activation induced cytine deaminase)
 In SHM : induced loss of amino group from
cytidine residues ,located in mutation hot
spots results in formation of uradile &this
creates u-G miss match & that creats of
shorter or longer stretches of mutated DNA.

37. In CSR:
Initiate the deaminating within the switch
region , upstream into both strand.

40.  T-dependent B-Cell Response
 primary immune response two sets of long-lived cells
remain:
 Memory B cells On secondary exposure to the same
antigen, the memory B cells will be stimulated and result
in production of high-affinity, heavy-chain class-
switched antibodies
Plasma cells
 Majority of the expanded population of antigen-specific B
cells undergo apoptosis

41.  Secrete antibodies, have few surface antibodies
 Those that arise from follicular B cells are found mainly in the
bone marrow and are long-lived (months)
 Those that arise from non-follicular B cells are short lived
(days to weeks)
 Plasma cell differentiation involves
 loss of Bcl6, Pax-5, CD19, CD20, and B cell activation
antigens
Appearance of XBP-1, BLIMP-1, CD38, CD138,
cytoplasmic Ig

42.  5-15 days after initial antigen exposure, a portion of the
germinal center B cells will upregulate IRF-4 (critical for
plasma cell differentiation).
 IRF-4 knockout mice lack Ig-secreting plasma cells
 IRF-4 over expression promotes plasma cell differentiation
 (IRF-4, BLIMP-1) down regulation of genes important for B
cell proliferation, class switching and somatic hypermutation
while upregulating synthetic rate of Ig synthesis and secretion
 Downregulation of CXCR5 (has kept the B cell in the germinal
center), upregulation of CXCR4 , leave the lymph node

43. Dendritic cell presents antigen
to developing B cells to see
which B cells are producing
antibody with high-affinity
for that antigen

47.  Genetic defects that prevent expression of the pre-B cell receptor or
that prohibit the transduction of signals via the receptor leads to
absence of B cells and agammaglobulinemia
 •Congenital agammaglobulinemia: loss-of-function mutations in
genes that encode components of the pre-B cell receptor or
downstream signaling molecules IgM
 Lambda 5 surrogate light chain
 CD79a, CD79b
 Bruton tyrosine kinase (BTK)
 B cell linker protein (BLNK)

48.  Characterized by markedly low serum IgG levels and low IgA
or IgM, poor/absent response to immunization
 Failed B cell differentiation with impaired secretion of
immunoglobulin
 reduced percentages of isotype switched memory B cells
 Variety of gene defects, most of which are sporadic

49.  Maturation of activated B cells in
absence of T cells
 Rapidly mature into short-lived plasma
cells without undergoing somatic
hypermutation or class switching
 Secrete IgM antibodies of low affinity
 Do not contribute to memory B cell
pools
 B-1 cells may preferentially follow this
non-follicular differentiation pathway
as they appear to be much less
dependent on T cell help for antibody
production