Activation of B cells and Production of Antibodies
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
Jason Cyster, PhD
Hemagglutininof influenza H1N1
Fab of broadly neutralizing antibody
Activation of B cells and Production of Antibodies
How is it that we can make an antibody against any foreign surface?
The life history of B lymphocytes
Newly produced B cells leave bone marrow and enter circulation
Migrate through secondary lymphoid organs and survey for antigens
Antigen-recognition; Interaction with helper T cells;
clonal expansion; (isotype switching)
Low-affinity Plasma cells-> Antibody
Germinal Center formation: somatic mutation and affinity maturation
High-affinity Plasma cells (->Antibody) and memory B cells
(continual)
(continual)
(a few days) (1-2 weeks)
(weeks)
(BCR)
-> Changes in gene expression include upregulation of B7
T cell
clonal expansion;differentiation
'activation' signalbut no clonal expansion
presentAg
T-independent (TI) T-cell dependent (TD)
Types of B cell Antigens: T-independent and T-dependent
clonal expansion;differentiation
BCR
Ag Ag Ag
• 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
• TCR triggering up-regulates 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)
CD4 T cell
CD40L (TNF family)
CD40 (TNF-R family)
TRAF2 TRAF3B cell
increased expression of cell cycle molecules, survival molecules,
promotes isotype switching
ICOS – ICOSL also required for Germinal Center responsesICOS 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
foreignprotein
sugar (polysaccharide)
Polysaccharide SpecificB cell
T
Foreign protein specific T cell
CD40LCytokines
BCR
MHC II
Ab
endosome
foreignpeptide
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
proteinhapten
Hapten SpecificB cell
+
1. Hapten covalently attaches to self-protein2. Hapten specific B cell binds haptenated-protein
3. Complex is internalized and degraded to haptenated peptides
4. Haptenated peptides are presented to T cells5. B cell receives help and secretes hapten specific
antibody
CD40LCytokines
T
Haptenated-peptide Specific
T cell
haptenatedself-peptide
Ab
membrane Ig
secretory Ig
B cell Plasma Cell
After appropriate activation the B cell differentiates into an antibody secreting cell (or Plasma Cell)
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
membrane Ig (BCR)
CH tmcypolyA polyA
secretory Ig (Ab)
B cell Plasma Cell
VH
- 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
CHVH
naive B cell activated B cells
3-4 days 12 divisions
plasma cells
1 day differentiation
1 day104 Ab/cell/sec
antibodies
1 212 = 4,096 4,096 >1012
B cell antibody response -> clonal replication enters into a higher order upon plasma cell differentation
bacteria - possibly dividing every ~60 min5 days = 2120 divisions
(Note: the exact numbers are not important)
Ig Heavy chain class (isotype) switching
VDJ m g e a
55 kb
(cytokines, CD40L)T cell help
antigen
IgM+ naive B cell
IgG+ memory cell
IgGsecreting plasma cell
constantvariable
Why make the different Ig isotypes? -> We will discuss
antibody effector mechanisms in the next lecture
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
VH VL
CLCH1
CDR1 2 3
CH2
CH3
CDR1 2 3
Ag
Mutations are targeted to antigen binding region of antibody
CDR = complementarity determining region, also known as the hypervariable region (part of V domain that binds the antigen)
Ag
before
Affinity maturation improves the ‘fit’ of the antibody for the inducing antigen
after
Ag
- increasing the binding affinity
Affinity maturation and antibody responses
AID dependent mutator complex
DNA replicationerror
ATG ... GGC TAT GCT CAC CGT ...
V CH1
...GGC, CCT...
Met ... Gly Tyr Ala His Arg ... ...Gly, Pro...
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-> mutations are actively induced in the V-regions of the
antibody heavy and light chain genes
ATG ... GGC TAT GTT CAC CGT ...
Met ... Gly Tyr Val His Arg ...
T
Val
...GGC, CCT...
...Gly, Pro...
V CH1
Somatic mutation of Ig V region in GC B cell
-> 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)
mantle zone
GC light zone (bright green staining, FDCs)
GC dark zone
T zone
(red - cell cycle marker-high cells that are rapidly dividing GC B cells)
(naive B cells)
Germinal Center in Human Tonsil
Germinal Center Dynamics
B
B
B
B BB
B BB
BB
B
MØ
B T
MØ
F F
B
B
B
B
BT
T
T
B
B
2. Dark Zone-> GC B cells (blasts) undergo
proliferation and somatic mutation of Ig V genes
3. Light Zone-> GC B cells compete to bind antigen displayed on Follicular
Dendritic Cells (F) and to receive T cell help
- > selection occurs for cells with higher affinity BCR
- > cells that fail to bind antigen die and are engulfed by
macrophages (MØ)
1. SeedingGC seeded by low affinity B cells that bound
antigen and received T cell help
4. Differentiation & Exit-> high affinity (selected)
B cells differentiate into long-lived
plasma cells
and
memory B cells that exit the GC
MB
PC
antigen
Response takes 1-2 weeks
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