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Lecture 6

Antibody genes II
Mechanism of class switching
Regulation of class switching
Somatic mutation
T cell dependent antibody response
Affinity maturation
T independent antibody responses

Next time: Mid term test

Review session
Saturday Jan 22
4:00-6:50PM YORK 2722

The qualities of T cell dependent antibodies change with time

Low affinity
IgM class

High
affinity
IgG
class

IgM

VDJ joining here creates heavy chain variable region domain


Switching to these classes requires
DNA recombination and is distinct from
V(D)J recombination.

VD

Note: there are exons encoding the membrane and secreted


forms of each of the antibody heavy chains.

Antibody gene
One exon is assembled from separate pieces by DNA rearrangement
in immature lymphocytes

DNA
V
Nave B cell IgM, IgD
DNA
C heavy

Antigen stimulated B cell


DNA

*** **
Point mutations
are introduced

On the antibody H chain, other exons are swapped


in by a DNA rearrangement process distinct from
V(D)J recombination

Heavy chain switch changes effector function, not specificity

S
VDJ

S
C

Joining can occur anywhere within the large, repetitive


S-regions. There is no obvious conserved sequence motif.
Unlike V(D)J recombination, this gene splicing occurs
between, rather than within, coding sequences.
Like V(D)J recombination, targeting of elements for
rearrangement is correlated with prior sterile transcription

B-cells can change their immunoglobulin class by DNA


recombination. This is a distinct process from V(D)J recombination!

+IL4 and CD40L (from CD4 T cells)

Like V(D)J recombination, class switch recombination is regulated by


1) expression of a recombination machine
2) targeted accessibility mediated by nearby enhancers and promoters

Figure 7-14

Ig isotype class switching

Concepts in class switching


Switching to H chain classes IgG, IgA, and IgE involves
irreversible DNA recombination.
During this process the H chain variable region retains
the same antigen specificity.
Recombination occurs within S (switch) regions, which
are several kilobases in length and highly repetitive (i.e,
nothing like the recognition sequences for V(D)J
recombination).
Recombination is targeted by DNA "accessibility" that is
controlled by sterile transcription starting at the I-region.
I region transcription is regulated by short and long range
cytokines provided mainly by T cells.
The specificity of the cytokines determines the H-chain
class to be used, and hence, the effector function.

Somatic mutation
adds to diversity
(specificity)

Accumulation of Vregion point mutations


during the antibody
response.

Mutations that improve


affinity for antigen are
selected in a quasiDarwinian process
during the immune
response.

Somatic mutations are focussed on the variable region exon

Promoter

Enhancer elements

Different cells
carrying the
same V gene
were compared
for sequence
changes

From Gearhart and Bogenhagen PNAS 80:3439, 1983

Patricia J. Gearhart and Richard D. Wood

Clues from the pattern of somatic mutation


Strand bias, transitions>transversions,
bias vs pyrimidines (as assessed on coding strand).

To
From
A
G
C
T

A
68
20
14

G
46
0
14

% of observed

C
29
24
72

T
25
8
80
-

mutations

33
35
21
10

From Betz et al, PNAS 90:2385.

Honjo's bombshell:
Knocking out a single gene blocks both somatic
mutation and class switching
A gene specifically expressed in activated B
cells undergoing class switching was knocked
out.
Mutant has hyper IgM syndrome.
IgM is only antibody class present in blood.
After immunization antibody V-regions have no
sign of somatic mutation.

Activation Induced cytidine Deaminase (AID)


Mutant mouse has no hypermutation or H-chain class switch
Homologous to RNA editase, but can deaminate DNA

AID

U
UNG
uracil-DNA
glycosylase

Transcription creates transient single stranded DNA


which is the substrate of AID

Model to explain
how AID-induced
mutations do not
cause just C-to-T
changes.

Neuberger et al 2003

Mismatch repair in eukaryotes

Msh2,6 +/- linked to


hereditary nonpolyposis colon
cancer (HNPCC)

Martin and Scharff Nat. Rev. 2:605 (2002)

-If mutations are routinely removed from replicating


DNA, a process that prevents repair locally would target
mutation. If so, knockouts of DNA repair genes would
have little or no effect.
-If mutations are introduced by massive local DNA
damage, possibly needed to overwhelm the normal
repair mechanisms, then repair mutants would have
increased mutation rates in the targeted regions (near
assembled VDJs).
-Alternatively, DNA repair enzymes may be needed to
generate mutations.

While mutations in DNA mismatch repair lead to an overall


increase in point mutations in the genome, they suppress Vregion point mutation suggesting that the mismatch repair
pathway is actively used during somatic mutation.

Mismatches can intitiate "patch" repair that introduces further


point mutations by using error-prone DNA polymerases

DNA polymerases

These three error prone DNA


polymerases have been implicated in
V-region somatic mutation

AID generates a lesion which is processed in different ways by DNA


repair enzymes and cofactors for hypermutation and class switch.

AID

AID

AID

Point mutation

H-chain switch

Honjo et al 2004

Somatic Mutation Concepts


Surprisingly, the diversity generated by V(D)J
recombination is insufficient, and is supplemented by
somatic point mutation!
Somatic mutation is induced upon antigen activation
among mature B cells.
CD4 T cell help is required.
Mutation is localized to antibody V region.
Mutation is initiated by AID at cytidines by deamination
(C>U); other repair enzymes extend the mutation.
In class switching, AID appears to generate staggered
nicks in S-regions, leading to the recruitment of the
non-homologous end joining enzymes (only this part
of the mechanism is reminiscent of V(D)J
recombination).

Chickens make a
single, invariant
antibody by V(D)J
recombination.
This antibody is then
massively mutated by
the process of gene
conversion, in which
patches of 15-20 bp
from nearby pseudoV
genes are substituted.
This process also
requires AID, but is
processed by a
different constellation
of repair enzymes that
point mutation or class
switch.
Fig 4.6 (left)

Risks and benefits of DNA manipulation by the immune system

Generation of diversity
-RAG1/2 DNA breaks, hairpins
-Terminal nucleotidyl transferase untemplated nucleotide additions
-AID point mutations, error prone repair, DNA breaks
Drawback: These processes probably contribute to cancer
Many (lymphoid) tumors involve somatic mutation and translocation
associated with antigen receptor genes

Cell:cell interactions in a T cell dependent antibody response


Antigen-specific B cell takes up antigen and
presents peptide to antigen-specific T cell.

Signal 1:
Antigen
crosslinks
sIg, leading to
activation

Signal 2
T cell help through
CD40-ligand and
cytokines

How do rare
antigen-reactive
B and T cells
find each
other?

Antigen specific B cells move after signal 1 to the T cell


zone
This interaction leads to proliferation of B cells
near the B:T zone border

Germinal centers are prominent in lymph nodes and spleen


They are mainly composed of activated B cells interacting with
antigen-specific T cells and follicular dendritic cells
Class switch and somatic mutation occur here

B
T

Figure 7-9 part 1 of 2

Figure 7-10

Figure 7-11 part 1 of 2

B cell survival and


proliferation in the
germinal center is
highly antigendependent.
Cells that mutate
to lower affinity die
of programmed cell
death.
Higher affinity
cells have a
competitive
advantage.

Figure 4-17

T dependent responses generate class switched


plasma cells sIg+ memory B cells

Many vital
antibody
responses do
not require T
cells.
But these
have limited
memory,
somatic
mutation, and
class
switching.

Figure 7-6

Fetal derived
B-1 type B
cells appear to
be restricted to
T-independent
antibody
responses

Figure 4-12

B cell tumors
mimic their
normal
counterparts
and carry
irreversible
DNA
modifications
that aid
diagnosis.

Figure 4-19

Monoclonal
antibody gene
rearrangements
can be detected in
tumors by
Southern blotting.

Common translocation between Ig and an oncogene


in Burkitt's lymphoma.

Modern drug research and development (duration ~10 years):


random screening of millions of compounds in bioassay
to find initial candidates
refining candidates based on minor substitutions and selection
for improved affinity and specificity
toxicity and efficacy trials
The antibody response (duration ~14 days):
select a few thousand (or fewer) cells among millions of B cells
point mutate and select to develop highest affinities*
*Important for recognition of microbes that mutate rapidly
class switch to appropriate effector class
End result: a long lived, high affinity antibody in circulation
Antibody molecules now behave like preexisting innate immune
molecules, allowing an instantaneous response to the specific microbe.
Note: a high affinity antibody is effective at lower dose, increasing the
lifespan of memory

Summary of lecture 6
Molecular mechanisms of class switching and somatic
hypermutation. AID and DNA repair enzymes.
Mismatch repair system is involved in point mutation.
Double strand break repair is involved in class switch.
CD4 T cells are important for class switch regulation
and the initiation of somatic mutation.
The T cell dependent antibody response requires cell
migration to bring T and B cells together.
Somatic mutation happens in the germinal center.
Selection in the germinal center favors high affinity
cells.
Plasma cells and memory B cells are formed during the
T dependent antibody response.
Review session
Next time: Mid term test

Saturday Jan 22
4:00-6:50PM YORK 2722