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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), andsite-specific mutagenesis that allows the selection of high-affinity 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
Figure 5.20
• 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
e.g.,IgE
-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
Figure 5.16
• We return to effector functions of antibodies in Ch.10• How do you switch from IgM/IgD to other isotypes?
Heavy chain constant region defines the isotype
Figure 5.3 + 5.16 + 5.18
• Not to scale and simplified• Figure above only shows protein-
coding regions. Regulatory elements in DNA provide control of recombination and transcription
Figure 5.3 + 5.16 + 5.18 + 5.25
L
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.
RAG-1 & -2 NOT required
Ig Class Switch Recombination (CSR):
L
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):
Extra-chromosomal DNAis lost/degraded
Class switching does not alter the VDJ sequence and consequently has no effect on antibody specificity.
Ig Class Switch Recombination (CSR):
Ig Class Switch Recombination (CSR) is instructed
Unlike V(D)J, which is largely random
CSR is initiated by CD40L:CD40 + appropriate cytokine
IgG1 IgG2a IgG2b IgG3 IgA IgE
IL-4 + +IL-5 +IFN + +TGF + +
Extra-chromosomal DNAis lost/degraded
IL-4 receptor
“sterile” transcription
Ig Class Switch Recombination (CSR):
• CSR requires:– Activation-induced deoxycytidine deaminase
(AID)– Uracil DNA glycosylase (UNG)
Ig Class Switch Recombination (CSR):
Properties of AID
AID
• Expressed mainly in GC B cells• Acts on single-stranded DNA
• AID converts C U at switch sites• U removed by UNG, “abasic site”• Cut by endonuclease• Joining to another switch site
Ig Class Switch Recombination (CSR):
Figure 5.25
“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
Ig Class Switch Recombination (CSR):
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):
Somatic Hypermutation (SHM):
Figure 5.24
Monoclonal antibodies as drugs…
Evidence-
• AID-/- mice & humans • UNG knockout mice
• Somatic hypermutation uses a similar mechanism
Somatic Hypermutation (SHM):The Germinal Center Reaction:
No CSRNO SHM
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.)
Im is the promoter forsterile (noncoding) transcription of Cm switch region
VDJ
Somatic hypermutation (SHM):
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
DNAreplication
•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
Somatic hypermutation (SHM):
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.
Somatic Hypermutation (SHM):The Germinal Center Reaction:
Evidence for a role of AID in SH and CSR:•AID-/- mice & humans: no SH or CSR•Fibroblasts transfected with AID gene &
substrate SHM & CSROther B cell-specific functions not
required (necessary and sufficient)
Figure 5.26
H L
H – heavy chain locusL – k and l light chain loci
H L
H L
H
H L
H
H
RAG-1/2
AID
AID
The brick?
The brick? AID
The brick? AIDThe window?
The brick? AIDThe window? Ig loci (light chain V region, heavy V and S)
The brick? AIDThe window? Ig loci (light chain V region, heavy V and S)Al?
The brick? AIDThe 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?
