Download Antibody Structure and Function

B Cell Generation, Activation,
And Differentiation
W. Robert Fleischmann, Ph.D.
Department of Urologic Surgery
University of Minnesota Medical School
[email protected]
(612) 626-5034
Objectives
• To understand B cell development
• To understand the rearrangement of the
antibody variable region during B cell
maturation
• To understand B cell activation and the
signals involved in the activation
process
• To understand the importance of
somatic cell mutations
• To understand antibody class switching
Harry Hammels, age 18 months, is
brought to the clinic by his parents. He
has fever (103.3°F), dyspnea, and nonproductive cough.
Harry has a history of repeated bacterial
upper respiratory infections.
What do you think?
What would you do next?
Total WBC count is 11,000 cells/µl
Differential count:
Neutrophils
40%
22-40%
Lymphocytes
45%
49-73%
Monocytes
5%
3-10%
Eosinophils
10%
0-6%
Basophils
0%
0-3%
What do you think?
What would you do next?
Oscultation: fine crepitant rales and rhonchi with focal
decreased resonance and bronchial breathing.
Bronchial breathing is indicative of
Consolidation
Cavitation
Complete alveolar atelectasis (collapse) with
patent airways
Mass interposed between chest wall and large
airways
Tension pneumothorax
Massive pleural effusion with complete atelectasis
of lung
Chest X-ray shows diffuse, bilateral perihilar
infiltrates.
What do you think?
What would you do next?
The clinical picture is indicative of an infection
with Pneumocystis jirovecii.
Examination of material from bronchial lavage
shows the presence of cysts, consistent with
Pneumocystis jirovecii.
What do you think?
What would you do next?
Lymphocytes are differentially identified.
CD4+ T cells: normal level
CD8+ T cells: normal level
NK cells: normal level
B cells: normal level
HIV tests:
ELISA test for HIV: negative
What do you think?
What would you do next?
B Cell Generation
B Cell Generation
• Progenitor B cells (pro-B cells) express c-Kit
which binds to stem cell factor expressed on
BM stromal cells, inducing them to proliferate
and differentiate to Precursor B cells (pre-B
cells).
• Pre-B cells express IL-7R and are stimulated
to divide and differentiate.
• Pre-B cells rearrange antibody  chain genes
first, then  chain genes.
B Cell Development
• Progenitor B cell
– cKit expressed/binds stem cell
factor
– Ig-/Ig- expressed
– Heavy chain gene
rearrangement
• Precursor B cell
– IL-7R expressed
– Express pre-B cell receptor
(heavy chain + surrogate light
chain)
– Light chain gene rearrangement
• Immature B cell
– IgM mRNA
– mIgM on cell surface
• Mature, naïve B cell
– Pre-mRNA differentially spliced
to IgM and IgD mRNAs
– mIgM/mIgD on cell surface
B Cell Generation
Surface
Markers
Intracellular Events
Progenitor B cell
cKit
Precursor B cell
IL-7R
Light chain gene
rearrangement
Immature B cell
mIgM
IgM mRNA
Mature, naïve B cell
mIgM/mIgD
Heavy chain gene
rearrangement
Pre-mRNA
differentially spliced
to IgM and IgD
mRNAs
Sequential Expression of
Membrane Immunoglobulin
B-1 B cells
•
•
A subset of B cells that is made
before the major group of B cells
(B-2 B cells)
Comprise 5% of total B cell
population
–
•
B-1 B cells exhibit a limited
repertoire of Ab variable regions
and the variable regions bind
antigen with lower affinity.
–
–
–
–
•
Mostly found in peritoneal and
pleural cavities where they
predominate
More likely to respond to
carbohydrate than to protein Ag
Ab are somewhat multi-specific
and can bind several Ags
Class switching is not common,
mostly IgM
No hypermutation of Ig genes, so
no affinity maturation
Unlike B-2 B cells, B-1 B cells are
self-renewing and can generate
more naïve B-1 cells.
Negative Selection of SelfReactive B Cells
• Negative selection of many self-reactive B cells
occurs in the bone marrow (clonal deletion).
– This limits development of antibody-mediated autoimmunity.
– If immature B cells, expressing mIgM recognize self-antigen,
• Some of the immature B cells undergo apoptosis.
• Some of the immature B cells undergo editing of light chain
genes to produce a different light chain that, when combined
with the heavy chain does not recognize self-antigen.
• Negative selection of some self-reactive B cells
occurs in the periphery.
– Not all self-reactive B cells are eliminated in the bone
marrow because not every self-antigen is expressed in the
bone marrow.
– Mechanism not really understood.
B Cell Activation by Antigen
Thymus-Dependent vs.
Thymus-Independent Antigens
• Thymus-dependent antigens (TD Ags)
require direct contact of B2 cells with Th2
cells to induce an antibody response.
– Generally, a stronger response than a thymusindependent Ag response
• Thymus-independent antigens (TI Ags)
induce B1 cells to produce antibodies without
the need for Th2 cell activity.
– Type 1 TI antigen = lipopolysaccharide and other
bacterial cell wall components
– Type 2 TI antigen = highly repetitious molecules
such as polymeric proteins (flagellin) or bacterial
cell wall polysaccharides with repeated subunits
B Cell Response to
Type 1 TI Antigen
• B1 cells bind lipopolysaccharide via either toll-like
receptor-4 (TLR4) or via the B cell receptor (specific
antibody + Ig- and Ig-).
– TLR4 binding is non-specific, so B cells with many different
antibodies will be activated (polyclonal activation). In this
way, it acts as a B cell mitogen.
– Antibody binding is specific, so only B cells with specificity
for the type of LPS will be activated.
• Type 1 TI antigen can activate both immature and
mature B cells because it doesn’t require a surface
antibody molecule (can activate through TLR4).
• Only IgM is produced.
B Cell Response to
Type 2 TI Antigen
• B1 cells bind Type 2 TI antigen by crosslinking
of the B cell receptor (specific antibody + Ig-
and Ig-).
– Antibody binding is specific, so only B1 cells with
specificity for the type of antigen will be activated.
– Only mature (not immature B1 cells) will be
stimulated.
• Mostly IgM is produced.
• While Th2 activity is not required, cytokine
production by Th2 cells are needed for full B
cell response and for class switching to
isotypes other than IgM.
Two Signals Are Required for
B Cell Activation
• TI Antigen
– Binding of antigen
provides both signal
1 and signal 2
• TD Antigen
– Binding of antigen
provides signal 1
– Binding of Th2 cell
via CD40:CD40L
provides signal 2
(analogous to B7 for
T cells)
Role of Ig-/Ig- in
Signaling
• mIgM and mIgD have short
cytoplasmic tails that are too short
to transduce a transmembrane
signal.
• Ig-/Ig- has ITAMs
(immunoreceptor tyrosine-based
activation motif) on the
cytoplasmic tails.
• The tyrosine based activation
motif is activated by antigen
cross-linking antibody on the
surface of the B cell.
• This initiates a cascade of events.
Intracellular
Activation
Cascade
• Sequential
phosphorylations
activate a series of
proteins that activate
phospholipase C that
in turn leads to
activation of
transcription factor
NF-B.
• Activation of B cell
receptor activates G
proteins that activate
transcription factors
Rho, Rac, and Ras.
• Stimulation
– B cell coreceptor
complex
– CR2 (CD21) binds to
complement C3d
bound to antigen to
cause
phosphorylation of
cytoplasmic tail of
CD19, initiating
further activation.
• Inhibition
– CD22
– Activates a
phosphatase that
cleaves phosphate
from ITAM.
– Blocks ITAM
signaling.
– CD22 KO mice
develop
autoimmunity.
Additional Regulation
of Signaling
B Cell Activation by T Cell
B Cell Activation by T Cell
• The activation of B cells by antigen
binding to receptor and by coreceptor
binding creates conditions for
proliferation and differentiation of B
cells.
• However, important T cell interactions
and T cell produced cytokines are
necessary to complete the activation of
B cells for thymus-dependent antigens.
Activation of B Cell by T Cell Requires Two Steps
•
•
The first signal is sent by antigen
binding.
The B cell takes up antigen by
receptor-mediated endocytosis.
– After about 30-60 minutes, the
antigen reappears on the B cell
surface bound to MHC
class II.
– The Th2 cell recognizes the MHC
class II bound antigen and, when
costimulated by B7 binding to
CD28, is activated.
•
The activated Th2 cell synthesizes
several molecules.
– CD40L = ligand for CD40 on B cell
causes the second signal for
proliferation to be sent
– Cytokines = IL-2, IL-4, and IL-5
push B cell to proliferate and
differentiate
•
The B cell expresses cytokine
receptors.
Primary vs. Secondary Immune Response
Comparison of Primary vs.
Secondary Immune Response
Lymph Node
Germinal Centers
•
•
•
•
•
•
•
Antigen flows through the lymph to
the lymph node either as free
antigen or as antigen bound to an
antigen-presenting cell.
Free antigen binds to follicular
dendritic cells in follicles and
germinal centers, macrophages
throughout the lymph node,
dendritic cells in the paracortex, or
B cells in the follicles and germinal
centers.
Processed antigen is presented to T
cells and to B cells.
T and B cells recognize each other
at the periphery of the medullary
region, bind, and move to the
germinal centers.
In the germinal centers, the B cells
known as centrocytes undergo
affinity maturation that is mediated
by somatic cell mutation.
In the germinal centers, there is
class switching.
The B cells become plasma cells
and exit the germinal centers.
Coordinant Movement of Bound B and T Cells
Importance of Somatic Cell Mutations
• Somatic hypermutation occurs within the VDJ
region with each cell division an activated B
cell.
• The hypermutations give rise to a slightly
different antibody molecule that may or may
not have greater affinity for the antigen
(affinity maturation).
– If less affinity, then antibody will not be able to
remain bound to antigen and apoptosis will
eliminate the B cell from the population.
– If greater affinity, the stronger interaction of the
antigen for the antibody drives proliferation, until a
clone of B cells with the greatest antigen affinity is
founded.
Class Switching
• An mIgM-bearing cell can produce secreted IgM or it can
undergo class switching by recombining the DNA at class switch
recombination sites.
• Class switching exchanges the constant region of the antibody
molecule .
– IgM becomes IgG
– IgG becomes IgE (recent work suggests IgM becomes IgE)
– IgG becomes IgA (recent work suggests IgM becomes IgA)
• Class switching is driven by B/T cell interactions via the
CD40/CD40L interaction and by specific cytokines produced by
the T cell.
• Class switching occurs in the germinal centers of lymph nodes.
• Individuals whose T cells lack CD40L cannot undergo class
switch and express a condition called X-linked hyper-IgM
syndrome.
– IgM only
– No memory cell generation
Harry Hammels
Harry is diagnosed with pneumonia
caused by Pneumocystis jirovecii. He
does not appear to be infected with HIV.
His T cells and B cells respond to
mitogen stimulation.
Antibody expression is evaluated:
IgM: higher than normal levels
IgG: absent
IgA: absent
What do you think is wrong with Harry?
Harry Hammels
It appears that Harry may have X-linked
hyper-IgM syndrome.
X-linked hyper-IgM syndrome occurs
because there is a failure of T cells to
send an appropriate signal to B cells for
them to class-switch.
Harry Hammels
Harry is found to lack expression of
CD40 ligand.
Without the binding of CD40 ligand on T
cells to CD40 on B cells, the B cells
cannot undergo class-switch. Hence,
hyper-IgM syndrome.
Differentiation and Class Switching Is
Also Driven by Specific Cytokines
Immunoglobulin Production DownRegulates the Immune Response
• As antibody is produced, it exerts a feedback inhibition
of its own production.
• This occurs by two mechanisms
– Soluble antibody binds antigen and prevents the antigen from
reaching the mIgM/mIgD antibody on the surface of other
naïve B cells.
– Antibody/antigen complexes bind to Fc receptor molecules on
the surface of the B cells making it ever more likely that the
down-regulatory events mediated by CD22 will be triggered.
• There are few molecules of CD22 on a cell and many molecules
of antibody.
• When few antibodies are bound to antigen, it is difficult for the
antigen/antibody complex and CD22 to find each other to bind.
• When many antibodies are bound to antigen, the
antigen/antibody complex and CD22 find each other easily.