Lecture 6
Development of
B Cells
“Most of what we once
thought we knew about global health has been proved wrong by the relentless
advances of HIV/AIDS, tuberculosis and malaria…..There can be no more urgent
cause facing us today. In
I.
B-Cell Maturation - review figure 5-1, page 112
This is the process that turns a hematopoietic stem cell into a naïve, but
antigen-responsive, B cell, figure 11-1, page 272.
A. Initial Steps (Bone Marrow)
1. Stem cell commits to
lymphoid line.
2. Lymphoid progenitor commits
to progenitor B cell, or pro-B cell, which develops a tyrosine receptor called
CD45R and has Iga/Igb transmembrane signal (figure 4-22, page 102)
3. Pro-B cells begin gene
rearrangement and differentiate into pre-B cells upon stimulation by the stromal
cells, figure 11-2, page 273.
B. Differentiation and Gene
Rearrangement (bone marrow), figure 11-3, page 274.
1. Pro-B cells bring DH
and JH together, figure 5-5, page 118.
2. Then they add leader plus VH
to DHJH, and do the P and N nucleotide additions.
3. If this produces a
non-productive (frame shifted) gene rearrangement, then they try the other
allele.
4. If the rearrangement is
productive, then the heavy chain is put into the membrane with a surrogate
light chain, composed of the products of two genes that can function without
rearrangement (figure 11-4, page 275).
5. The immature receptor
associates with the Iga/Igb transmembrane signal (figure 4-22, page 102).
6. Once there is a productive H
chain gene, the cell is a pre-B cell. If
there is no productive rearrangement, the cell apoptoses.
7. The pre-B cell then
undergoes rearrangements of first one k, then the other, and one l, then the other, stopping as soon as there
is a productive light chain arrangement and ultimately apoptosing if there is
not.
8. Once you have two productive
rearrangements, you have an immature B cell, one that has a determined
antigenic specificity (CDR) and uses the m CH region to
produce membrane-bound antibody.
9. Shortly thereafter, the cell
also begins to express membrane-bound d CH, and become a
mature (but naïve) B cell, expressing IgM and IgD on the cell surface.
10. At this point the cells are
released into the plasma and head to the peripheral (secondary) lymphoid
organs: lymph nodes, spleen and mucosa.
C. Removing Self-Reactive B
Cells.
1. 90% of the B cells produced
by the above process never make it to the plasma.
2. The elimination is signaled
by the crosslinking of IgM.
3. Artificially crosslinking IgM will lead to apoptosis of developing B
cells.
4. However, B cells can
sometimes get a second chance.
5. If they have a k chain involved in the CDR for a
self-antigen, they may try to replace it with a l. If the new CDR does not recognize a
self-antigen, then the cell is rescued and can enter the plasma.
II. B-Cell Activation and
Proliferation
A. Antigen Exposure, figure
11-9, page 280
1. If a circulating B cell does
not encounter an antigen that can bind to its surface antibody, it will undergo
apoptosis and die within a few weeks.
2. Thymus independent (TI)
antigens are those that can activate a B cell directly. These antigens also
simultaneously activate toll-like receptors.
a. Type 1 antigen -
lipopolysaccharide such as those found in the outer bacterial cell walls of
gram negative bacteria, which also activates TLR4
b. Type 2 - repetitive
polymeric proteins, such as bacterial flagellin, can cross link the
membrane-bound immunoglobulins and kick off proliferation.
3. However, this process does
not induce class switching (you mostly just make IgM) and does not produce
memory cells. For that you need TH
cells.
B. Activating Signals
1. Recall that while a mIgM or
mIgD molecule can bind antibody, it doesn’t stick far enough into the cell to
transduce this as a signal to the interior, figure 11-8, page 280.
2. The cytoplasmic tails of the
Iga/Igb stick into the cell and
participate in a tyrosine kinase signaling pathway. The tails of the molecules constitute the immunoreceptor tyrosine-based activation
motif, or ITAM.
3. When an antigen cross-links
one antibody with the next outside the cell, it brings together the internal
receptor molecules.
4. This causes the complex to
change conformation and recruits src (the original enzymes were isolated from
cells infected with Rous sarcoma virus) tyrosine kinases (blk, lyn, fyn) that
add phosphates to the tyrosines of the
5. This triggers multiple internal
signaling pathways that lead to changes in gene expression, the transcription
factor NF-κB being involved in this.
The is the same transcription factor activated by toll-like receptors, figure
11-10 page 282
6. CD-22 signaling provides
brakes on the system
C. Role of TH cells,
figure 11-12, page 285
1. However, the
2. When B cells bind antigen,
they bring some it inside and hydrolyze it.
3. Some of the hydrolyzed
peptide winds up attached to class II MHC molecules, the genes for which are
upregulated along with the one for B7.
4. Thus the B cell can present
some of the antigen to a TH cell and also contact the T cell using
B7 to CD28.
5. Te cells attach, forming a
conjugate or immune synapse, figure 11-13, page 286.
6. This causes the TH
cell to produce CD40L, which is a juxtacrine factor that turns around and
signals the B cell through CD40 receptor.
7. The contact reorganizes the
interior of the TH cell so that cytokines are released toward the B
cell.
8. The B cells begin producing
receptors for the cytokines.
9. Cytokine signaling activates
the B cells and they begin proliferating and differentiating.
III. Primary Versus Secondary
Response, Table 11-4, page 290, figure 11-16, page 289
A. The Primary Response
1. naïve lymphocytes
2. 4 to 7 day lag time
3. produces antibody secreting
plasma cells and memory cells
4. initial antibodies mostly
IgM; IgG toward the end
B. The Secondary Response: The
Sadder, but Wiser, Immune System
1. Produced primarily by memory
cells
2. 1 to 3 day lag time
a. The number of memory cells
specific for the antigen increases.
b. These memory cells are more
easily activated.
c. They have already been
through affinity maturation, so they're better at binding antigen
3. more antibody secreted, and
over a longer time
4. much higher proportion of
IgG and other isotypes
IV. B-Cell Maturation in
Anatomical and Histological Context
A. Lymph Nodes, figure 11-17,
page 292 - primary site for B cell maturation.
1. Lymph drains from tissues
and passes through these.
2. Antigen enters. It can be
a. "free" - particle
from pathogen, or the whole bacteria or viruses themselves
b. proteins or other antigens
from the pathogens complexed with antibodies
c. carried in by presenting
cell (dendritic or macrophages) that have picked it up elsewhere
3. Free and antibody-bound
antigen in the plasma is likely to be picked up by
a. interdigitating dendritic
cells
b. macrophages,
c. follicular dendritic cells
4. Naïve lymphocytes from the
bone marrow enter via the lymph.
5. Activation begins in the
paracortex, the layer between the outer cortex and the inner medulla, where
there is a high concentration of T cells, macrophages, and dendritic cells.
6. First the macrophages and
the dendritic cells activate the TH cells.
7. Naïve B cells contact the TH
cells, presenting any antigen they have internalized via the class II
8. The B cell begins to divide,
producing a clonal cluster (focus) at the boundary between the paracortex and
cortex.
9. A few activated B and TH
cells migrate together from one of these foci to a primary follicle in the
cortex.
10. The follicle becomes a
secondary follicle, one with a germinal center where B, TH, and
follicular dendritic cells interact, figure 2-15, page 44
11. A reminder about follicular
dendritic cells: these are NOT
regular sentinel dendritic cells..
B. Germinal Centers (figure
11-20, page 294)
1. Activated B cells
(centroblasts) proliferate and move to one edge or the follicle, forming a dark
zone. At this stage the centroblasts:
a. enlarge and begun to divide
rapidly
b. begin somatic hypermutation-
mutating the regions in the heavy and light chain genes that code for the
variable loops.
c. stop displaying the membrane
Ig (recycles the original via membrane turnover)
2. The centroblasts
differentiate into centrocytes which
a. stop dividing
b. begin expressing membrane Ig
c. move into light zone
d. contact follicular dendritic
cells
e. undergo selection B cells
with more effective receptors will survive and multiply at a greater rate,
out-competing those B cells with antibody receptors less capable of competing
for antigen-antibody complexes.
3. What happens in the
follicles is a highly unusual form of accelerated natural selection.
a. random mutation (dark zone)
b. excess reproduction (dark
zone)
c. selection (light zone) -
4. The centrocytes leave the
germinal center as plasma cells, lose their surface antibody again, and begin
secreting antibodies.
5. Most centrocytes do not
contact an antigen that fits with their surface receptors, however, and these
die by apoptosis, and are recycle by macrophages.
6. Purpose - The environment of the light zone selects
for those centrocytes that express the most effective antibodies.
a. The maturing B cell that
enters the germinal center and begins dividing does so if it can bind (with its
surface antibody) to some degree an antigen currently arriving in the lymph
node.
b. It differentiates into a
centroblast that undergoes random mutational events to the very region of the
gene responsible for determining the effectiveness of this antigen binding (the
CDR).
c. The mutated centrocyte now
displays the new antibody at its surface.
d. As with most random
mutations, most of the progeny centrocyte cells produced by this will bind
antigen less effectively than the original cell.
e. A few however, will bind the
antigen more effectively. These will
compete more effectively in the next stage for the antigen-antibody beads
supplied by the follicular dendritic cells.
Cells with improved receptors divide more rapidly than cells with less
effective receptors.
7. Signals
a. Follicular dendritic cells
play an important role in the selection process. The (iccosome)
beads are essentially a scarce resource, and the centrocytes have to compete
for them.
b. Thus the more effectively
the centrocyte surface antigen binds to the antibody displayed by the
follicular dendritic cell, the more likely it is to live.
c. In addition, the centrocytes
have to receive signals from the TH cells, especially the contact of
CDC40L to the CDC40.
C. Class Switching – directed
by TH cells, figure 11-22, page 296
1. The next decision the future
plasma cell must make is exactly what class of antibody to send out with the
refined CDR region produced by affinity maturation.
2. Cytokine signals from the TH
cells will determine this (more later).
D. To Remember or to Act: The
Final Decision.
1. Centrocyte now decides
whether to become a plasmoblast and generate a plasma cell or become a memory
cell and wait for a subsequent exposure to antigen.
2. Recall that plasma cells do
not express membrane-bound antibody. This means that the sequence of
differentiation in the lymph node involves:
a. dividing mature B cells with
surface antigen
b. dividing centroblasts with no
surface antigen (undergoing hypermutation)
c. non-dividing centrocytes
expressing surface antibody and undergoing selection
d. dividing plasma cells not
expressing surface antibody secreting soluble (humoral) antibody
3. The final differentiation to
a plasma cell involves the switch that generates the splicing enzymes that do
not add the membrane spanning exons to the mH chain message.
4. Also transcription and
translation levels generally rise as the cell begins cranking out antibody, as
does the proportion of RER.
5. Memory cells set aside from
this process may resemble naïve B cells, but they have undergone class
switching and make a variety of heavy chains, Table 11-6, page 297.
6. The receptors of memory
cells may therefore also be membrane-bound versions of IgG, IgA, and IgE.
V. Regulation
A. B-Cell Differentiation
1. B-Cell Specific Activator
Protein (BSAP) functions as master regulator.
2. Present ONLY in members of the B cell lineage.
3. Present in ALL members of the B cell lineage EXCEPT mature plasma cells, which are
done differentiating.
4. Binds to a variety of B-cell
gene promoter regions, including those like the surrogate L chains and class
switching regions that are involved in developmental decision making.
5. High levels tend to maintain
a cell as a memory cell, low levels tend to promote formation of plasma cells.
B. Overall Immune Effector
Response - Tolerance
1. You would like NOT to make antibodies against your own
proteins, and therefore tolerate them.
2. On the other hand, you do NOT want to develop tolerance for
foreign antigens, especially those associated with pathogenic infection.
3. Constant monitoring of your
antigens by Treg cells suppresses immune responses to your own
proteins and to those of benign commensal bacteria and fungi.
4. Moreover, you need to apply
brakes once an infection is under control
5. If you introduce foreign
antibodies to an antigen, this will tie up the antigen and prevent it from
promoting an immune response on the part of the host:
a. measles-mumps-rubella after
1 year
b. biological warfare
B-lineage Development
Summary
|
name |
location |
Surface receptor molecule |
Other surface signaling molecules |
activity |
|
Pro B cell |
Bone marrow |
Igα/Igβ and co-receptor |
C-kit, CD45R, VLA-4. IL-7 receptor, CD19, CD 43 |
Rearranging heavy chain gene |
|
Pre B |
Bone marrow |
Heavy chain plus surrogate light chain and co-receptor |
CD45R, VLA-4. IL-7 receptor, CD25 (α chain of
IL-2 receptor) |
Rearranging heavy gene |
|
Immature B cell |
Bone marrow |
mIgM and co-receptor |
No CD 25 |
Undergoing selection against self-recognition, changing
RNA processing |
|
Mature, naïve B cell |
Circulates: plasma, secondary and tertiary
lymphoid tissue |
mIgM and mIgD and co-receptor |
|
Trolling for antigen. Antigen binding and activation necessary
for next stage |
|
Activated B cell - 1 |
Peripheral lymphoid tissue (lymph nodes as
example) at paracortex |
mIgM and mIgD and co-receptor |
|
Begins clonal expansion |
|
Activated B cell - 2 |
Cortex, primary follicle, which then become
secondary, with germinal center |
mIgM and mIgD and co-receptor |
Up-regulate MHCII, CD 40, various cytokine
receptors |
Associate with T cells |
|
centroblast |
secondary follicle, periphery (dark zone) of the
germinal center |
No surface Ig receptor |
|
Divide rapidly, mutate Ig genes at specific
regions of CDR loops |
|
centrocyte - 1 |
Cortex, now
secondary follicle, periphery, light zone |
altered mIgM and mIgD and co-receptor |
CD40 (sign from CD40L necessary, too) |
Stop division, compete for antigen displayed on
follicular dendritic cells. |
|
centrocyte - 2 |
Cortex, now
secondary follicle, periphery, light zone |
altered mIgM and mIgD and co-receptor |
CD40 (sign from CD40L necessary, too) |
Decide whether or not to class switch and whether
or not to become a memory cell |
|
plasmablast |
Lymph node |
none |
|
Switch to non-membrane spanning heavy chain and
up-regulation of Ig synthesis |
|
plasma cell |
Circulation, site of infection |
none |
|
Secretes antibody |
Connections:
1. Properties of antigen that
affect binding to antibodies, Lecture 4, III
2. Antigen-presenting cells and
follicular dendritic cells, Lecture 2, II
3. Lymph nodes and spleen,
Lecture 2, V
4. Generation of antibody diversity,
Lecture 5, II
5. Autoimmunity, Lecture 16
6. TH cell cytokine
signaling, Lecture 11, III
7. Hypersensitivity (Allergy), Lecture
15
8. Passive Immunity and
Vaccines, Lecture 19
References and Links: