Activation of B cells and production of Antibodies

Antigen

Antibody 1

Antibody 3

Antibody 2

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)

• T-dependent antigens must contain a protein component (true of most antigens)

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'

CD4 T cell

CD40L (TNF family)

CD40 (TNF-R family)

TRAF2 TRAF3B cell

increased expression of cell cycle molecules, survival molecules,

promotes isotype switching

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

Protein SpecificT cell

CD40LCytokinesBCR

MHC II

endosome

Haptens and hypersensitivity reactions

• Small organic molecules of simple structure do not provoke antibodies by themselves. However, antibodies can be raised against them if the molecule is 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

• urushiol in poison oak and the nickel in earrings that cause contact dermatitis are both haptens involved in T cell-mediated DTH 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

antibodyCD40LCytokines

T

Haptenated-peptide Specific

T cell

membrane Ig

secretory Ig

B cell Plasma Cell

After appropriate activation the B cell differentiates into an antibody secreting cell, also known as a 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

Ig Heavy chain class (isotype) switching

VDJ

55 kb

(cytokines, CD40L)T cell help

antigen

IgM+ naive B cell

IgG+ memory cell

IgGsecreting plasma cell

Affinity Maturation• Affinity maturation occurs in germinal centers

(GC) and is the result of somatic hypermutation of Ig-genes in dividing B cells followed by selection of high affinity B cells by antigen displayed by FDCs

• 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

T ...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

Val

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

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 FDC (F)

and to receive T cell help-> selection occurs for cells

with higher affinity BCR; these cells successfully compete to bind antigen, present peptide-MHC II complexes to T cells

and survive-> 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

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