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
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 an ITAM motif
(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
antibody 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
– IgG 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
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.