Chapter 4 and 5 Ig study questions (Tu):
• Can you name at least four ways in which CSR
and V(D)J recombination differ?
• What are the substrates (what genes, what
areas) for CSR and SHM? What are the key
factors?
• How does AID work? Where does it operate?
• How are specific class switch events regulated?
• Why are antibodies used so widely, as lab
reagents, diagnostics, therapeutics?
The brick?
The brick? AID
A difference between Ig and
TCR: TCR solely functions as
an antigen-specific receptor,
whereas Ig encodes both
antigen receptor and the major
effector molecule. Effector
functions are mediated by the
IgH constant regions.
Consequently, the
molecular biology of the
BCR/antibody
(immunoglobulin) gene
loci following activation
is much more complex
i.e., there are further
recombination events to
generate antibodies with
specific effector functions (Ig
isotype switching), and
site-specific mutagenesis that
allows the selection of highaffinity antibodies (affinity
maturation)
• Immunological phenomena:
-Isotype switching
-Affinity maturation
Happen after stimulation of
mature B cells, in the secondary
lymphoid organs (LN, spleen,
Peyer’s patches).
Mostly occur in germinal centers
(“the germinal center reaction”)
Figure 1.18
• Immunological phenomenon:
-Isotype switching
Molecular mechanism:
-Class switch recombination
• Immunological phenomenon:
-Affinity maturation
Molecular mechanism:
-Somatic hypermutation
e.g.,
IgE
• V segment generated by V(D)J recombination is fixed,
but can become mutated by SHM (heavy and light)
• Class switch recombination changes out heavy chain
constant regions, replacing IgM/IgD with a “switched”
isotype
Figure 5.20
-Isotype switching
•same Ag binding site
different heavy chain constant
regions and effector properties
•Ig comes from the same
(heavy and light chain) gene
loci
Heavy chain constant region defines the isotype
• We return to effector functions of antibodies in Ch.10
• How do you switch from IgM/IgD to other isotypes?
Figure 5.16
• Not to scale and simplified
• Figure above only shows proteincoding regions. Regulatory
elements in DNA provide control
of recombination and
transcription
Figure 5.3 + 5.16 + 5.18
L
Figure 5.3 + 5.16 + 5.18 + 5.25
Ig Class Switch Recombination (CSR):
When B cells leave the bone marrow, they express IgM and IgD on their
surface.
During an immune response, B cells may undergo “class switching” - to
give rise to B cells producing IgG, IgA or IgE.
L
RAG-1 & -2 NOT required
Ig Class Switch Recombination (CSR):
During class switching, recombination occurs between switch sites.
The initial recombination will occur between the m switch site and a downstream
switch site.
Since there is no switch site associated with the d constant region gene segment,
the B cell cannot class switch to IgD.
Ig Class Switch Recombination (CSR):
Class switching does not alter the VDJ sequence and
consequently has no effect on antibody specificity.
Extra-chromosomal DNA
is lost/degraded
Ig Class Switch Recombination (CSR) is instructed
Unlike V(D)J, which is largely random
CSR is initiated by CD40L:CD40 + appropriate cytokine
IgG1
IL-4
IgG2a
IgG2b
IgG3
+
TGF
IgE
+
IL-5
IFN
IgA
+
+
+
+
+
Ig Class Switch Recombination (CSR):
IL-4 receptor
“sterile” transcription
Extra-chromosomal DNA
is lost/degraded
Ig Class Switch Recombination (CSR):
• CSR requires:
– Activation-induced deoxycytidine deaminase
(AID)
– Uracil DNA glycosylase (UNG)
Properties of AID
•
•
Expressed mainly in GC B cells
Acts on single-stranded DNA
AID
Ig Class Switch Recombination (CSR):
•AID converts C  U at switch sites
•U removed by UNG, “abasic site”
•Cut by endonuclease
•Joining to another switch site
Figure 5.25
Ig Class Switch Recombination (CSR):
“sterile” transcription
Ig Class Switch Recombination (CSR):
germline transcript=
sterile transcript
Ig Class Switch Recombination (CSR):
Evidence• AID-/- mice & humans
• UNG knockout mice
No CSR
Somatic Hypermutation (SHM):
Occurs during antibody response - NOT B cell maturation.
During proliferation of B cells in germinal centers, there are
increased mutation rates in the sequence encoding the V
regions of the BCR (Ab).
This will randomly alter (increase or decrease) the affinity of
the B cell’s BCR (Ab) for the eliciting antigen.
Those B cells with increased affinity for the antigen are
selected.
As a result, as an immune response proceeds, the affinity of
antibody produced increases  “Affinity maturation”
Somatic Hypermutation (SHM):
Figure 5.24
Monoclonal antibodies as drugs…
The Germinal Center Reaction:
Somatic Hypermutation (SHM):
Evidence• AID-/- mice & humans
• UNG knockout mice
No CSR
NO SHM
• Somatic hypermutation uses a
similar mechanism
Somatic hypermutation (SHM):
Im is the promoter for
sterile (noncoding)
transcription of Cm switch
region
VDJ
From Martin & Scharff (2002)
•SHM targets V, not C region;  = 1/1000 bp/division
•Transcription means RNA polymerase, not DNA
polymerase (typo on page 182 of Janeway textbook.)
Properties of AID
•
•
Expressed only in GC B-cells
Acts on single-stranded DNA
Properties of AID
•
•
Expressed only in GC B-cells
Acts on single-stranded DNA
DNA
replication
Somatic hypermutation (SHM):
•AID converts C  U at Ig V exon
•U can be fixed as T by replication, or
•U removed by UNG, “abasic site”
•Cut by endonuclease
•Error-prone repair
Cryosection of lymph node obtained 5
days after secondary antigen
injection. Green: ki-67--dividing cells
(spatially defines the germinal center).
Red: IgM--primary B cell follicle.
Yellow: Macrophages.
The Germinal Center Reaction:
Somatic Hypermutation (SHM):
Evidence for a role of AID in SH and CSR:
•AID-/- mice & humans: no SH or CSR
•Fibroblasts transfected with AID gene &
substrate  SHM & CSR
Other B cell-specific functions not
required (necessary and sufficient)
RAG-1/2
H L
H L
H L
AID
H
AID
H L
H
H
H – heavy chain locus
L – k and l light chain loci
Figure 5.26
The brick?
The brick? AID
The brick? AID
The window?
The brick? AID
The window? Ig loci (light chain V region, heavy V and S)
The brick? AID
The window? Ig loci (light chain V region, heavy V and S)
Al?
The brick? AID
The window? Ig loci (light chain V region, heavy V and S)
Al? Natural Selection (APOBECs)
Chapter 4 and 5 Ig study questions (Tu):
• Can you name at least four ways in which CSR
and V(D)J recombination differ?
• What are the substrates (what genes, what
areas) for CSR and SHM? What are the key
factors?
• How does AID work? Where does it operate?
• How are specific class switch events regulated?
• Why are antibodies used so widely, as lab
reagents, diagnostics, therapeutics?