Activation of B cells and Production of Antibodies
Jason Cyster, PhD
Learning Objectives of lecture:
•
Describe the key changes that occur in the B cell upon binding antigen
•
Understand the 2 classes of antigen, T-independent & T-dependent
•
Explain what a B cell must do in order to receive T cell help
•
Describe the major components of T cell help and appreciate the
defects that can cause humoral immunodeficiency
•
Explain how a conjugate vaccine works and how you can make an
antibody response to a hapten
•
Explain how isotype switching occurs
•
Describe the properties of plasma cells
•
Describe the process of antibody affinity maturation in germinal
centers, recognizing the 2 key processes involved and the cellular
outputs
•
Understand the main Ig isotypes made during memory responses and
why these responses are faster and of greater magnitude
Activation of B cells and Production of Antibodies
Fab of
broadly
neutralizing
antibody
How is it that we
can make an
antibody against
any foreign
surface?
Hemagglutinin
of influenza H1N1
The life history of B lymphocytes
Newly produced B cells leave bone marrow and enter
circulation
(continual)
Migrate through secondary lymphoid organs and survey for
antigens
(continual)
Antigen-recognition; Interaction with helper T cells;
clonal expansion; (isotype switching)
(a few days)
Low-affinity Plasma cells
-> Antibody
(1-2 weeks)
Germinal Center formation: somatic
mutation and affinity maturation
(weeks)
High-affinity Plasma cells (->Antibody)
and memory B cells
(BCR)
-> Changes in gene expression include upregulation of B7
Types of B cell Antigens: T-independent and T-dependent
T-independent (TI)
T-cell dependent (TD)
Ag
Ag
Ag
T cell
present
Ag
BCR
clonal expansion;
differentiation
'activation' signal
but no clonal expansion
clonal expansion;
differentiation
• T-independent antigens are multivalent (e.g. bacterial polysaccharides
or repeating determinants on the surface of viruses)
– fast (within 1-2 days) and predominantly IgM
– weak in infants and young children
• T-dependent antigens must contain a protein component (true of most
antigens) so that T cell help can be received
– slower (initiate over several days), involve all Ig isotypes (IgM, IgG, IgA, IgE)
– can lead to antibody affinity maturation and memory
(T zone)
Antigen (red)
Specific B cell (green)
Antigen presentation by B lymphocytes to helper T cells
B cells present
antigen they are
specific for 100,000
times more efficiently
than a non-specific
antigen
Mechanisms of helper T cell-mediated activation
of B lymphocytes
Role of CD40 in B cell activation
CD4 T cell
CD40L (TNF family)
CD40 (TNF-R family)
B cell
TRAF2
TRAF3
increased expression of cell cycle
molecules, survival molecules,
promotes isotype switching
• TCR triggering upregulates CD40L on T cell
• CD40 signaling promotes B
cell activation, isotype
switching
• CD40 also important in DC,
Macrophage function
• CD40L-deficiency =
'hyper-IgM syndrome’
(X-linked)
ICOS – ICOSL also required for Germinal Center responses
ICOS deficiency is a cause of Common Variable Immunodeficiency (CVID)
Linked help and the
Conjugate Vaccine concept
• Many bacteria are heavily coated with surface
polysaccharides
• Vaccines against these bacteria aim to induce
antibodies specific for the polysaccharide e.g.
• Haemophilus influenzae Type b vaccine
• Pneumococcal vaccine
• Meningococcal vaccine
• But infants and young children mount poor
T-independent antibody responses
• Conjugate vaccines link the polysaccharide to an
immunogenic protein carrier so that a T-dependent
antibody response can be induced
Mounting a T-dependent antibody response to a
polysaccharide in a conjugate vaccine
foreign
protein
Ab
sugar (polysaccharide)
BCR
CD40L
Cytokines
T
MHC II
endosome
Polysaccharide Specific
B cell
foreign
peptide
Foreign protein
specific T cell
Haptens and hypersensitivity reactions
• Small organic molecules do not provoke
antibodies by themselves
• Antibodies can be raised against them if
attached to a protein carrier
• Termed haptens (from the Greek
haptein, to fasten)
• Some drugs (e.g. Penicillin) can act as
haptens and induce antibody-mediated
allergic reactions
Making an antibody response to a hapten
hapten protein
+
1.
2.
Hapten covalently attaches to self-protein
Hapten specific B cell binds haptenated-protein
3.
Complex is internalized and degraded to haptenated
peptides
Haptenated peptides are presented to T cells
B cell receives help and secretes hapten specific
antibody
4.
5.
Ab
CD40L
Cytokines
T
Hapten Specific
B cell
haptenated
self-peptide
Haptenatedpeptide Specific
T cell
After appropriate activation the B cell differentiates into an
antibody secreting cell (or Plasma Cell)
B cell
Plasma Cell
membrane Ig
secretory Ig
After their generation in secondary lymphoid organs, many Plasma Cells
home to the bone marrow or mucosal surfaces (or lactating mammary
gland) where they live for many months, continually secreting antibody
Production of membrane vs secreted Ig
B cell
Plasma Cell
membrane Ig (BCR)
secretory Ig (Ab)
VH CH
VH CH tmcy
polyA
polyA
- B cells express Ig Heavy chain transcripts that include transmembrane and
cytoplasmic domains
- Plasma cells express Ig Heavy chain transcripts that stop after the CH domains,
thereby encoding the same antibody but in a secreted form
B cell antibody response -> clonal replication enters into a
higher order upon plasma cell differentation
(Note: the exact numbers are not important)
3-4 days
12 divisions
naive
B cell
1
1 day
differentiation
activated B cells
212 = 4,096
1 day
104 Ab/cell/sec
plasma cells
4,096
bacteria - possibly dividing every ~60 min
5 days = 2120 divisions
antibodies
>1012
Ig Heavy chain class (isotype) switching
variable
VDJ
constant
m
g
e
a
55 kb
T cell help
(cytokines, CD40L)
antigen
IgM+ naive
B cell
Why make the different Ig isotypes?
-> We will discuss antibody effector
mechanisms in the next lecture
IgG+
memory
cell
IgG
secreting
plasma cell
Affinity Maturation
• Affinity maturation occurs in germinal centers
and is the result of (1) somatic hypermutation of
Ig-genes in dividing B cells followed by
(2) selection of B cells for their ability to bind more
strongly (with higher affinity) to the inducing
antigen
• The high affinity B cells emerging in germinal
centers give rise to long-lived plasma cells and
memory B cells
Mutations are targeted to antigen binding region
of antibody
Affinity maturation improves the ‘fit’ of
the antibody for the inducing antigen
Ag
CDR CDR
1 2 31 2 3
VH
CH1
VL
before
after
Ag
Ag
CL
- increasing the binding affinity
CH2
CH3
CDR = complementarity determining region, also known as the
hypervariable region (part of V domain that binds the antigen)
Affinity maturation and antibody responses
Somatic mutation of Ig V region in GC B cell
-> mutations are actively induced in the V-regions of the
antibody heavy and light chain genes
V
CH1
Met ... Gly Tyr Ala His Arg ...
...Gly, Pro...
...GGC, CCT...
ATG ... GGC TAT GCT CAC CGT ...
DNA replication
error
AID dependent
mutator
complex
AID = Activation Induced Deaminase
(-> deaminates Cytosine on Uracil
-> repair proteins then come in and this leads to error prone repair)
Somatic mutation of Ig V region in GC B cell
V
CH1
Met ... Gly Tyr Val His Arg ...
...Gly, Pro...
...GGC, CCT...
ATG ... GGC TAT GTT
T CAC CGT ...
-> now encodes antibody molecule with slightly altered antigen
binding site
-> sometimes, by chance, this site will have an improved ability
to bind the inducing antigen (i.e. a higher affinity)
Germinal Center in Human Tonsil
mantle zone
(naive B cells)
GC light zone
(bright green staining,
FDCs)
GC dark zone
(red - cell cycle marker-high
cells that are rapidly
dividing GC B cells)
T zone
Germinal Center Dynamics
3. Light Zone
-> GC B cells compete to bind
antigen displayed on Follicular
Dendritic Cells (F) and to
receive T cell help
antigen
B
T
B
F
B
F
T
-> selection occurs for cells
with higher affinity BCR
MØ
B
B
and
MB
memory B cells
that exit the GC
MØ
B
B
B
B
B
B
-> GC B cells (blasts) undergo
proliferation and
somatic mutation of Ig V genes
-> high affinity (selected)
B cells differentiate into
long-lived
PC plasma cells
B
B
2. Dark Zone
T
B
B
B
-> cells that fail to bind antigen
die and are engulfed by
macrophages (MØ)
4. Differentiation & Exit
B
Response takes
1-2 weeks
B
B
B
T
1. Seeding
GC seeded by low affinity B cells that bound
antigen and received T cell help
Memory B cells
• Generated in germinal centers
– therefore we only have strong humoral memory to T-dependent
antigens
•
•
•
•
Small, recirculating cells
Often isotype switched (e.g. IgG+ or IgA+)
Typically have higher affinity for the inducing Ag
Longer lived than naïve B cells
– Persistence of memory B cells after an immune response
ensures that we have increased numbers of B cells specific for
the antigen and ready to respond on re-encounter
Features of primary and secondary antibody responses