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