The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 1
1
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The Medical Research Council
The Laboratory of Molecular Biology
University of Cambridge,
U.K
2
Thucydides c.429 BC – the plague of Athens
(Slide courtesy J. Sale)
………for the same man was never attacked twice –
never at least fatally, and such persons not only received
the congratulations of others, but themselves also,
in the elation of the moment,
half entertained the vain hope that they were for the future safe
from any disease whatsoever
3
Letter XI - on inoculation
It is inadvertently affirmed in the Christian countries
of Europe that the English are fools and madmen;
Fools, because they give their children the smallpox
to prevent them catching it; and madmen, because they want
only communicate a certain and dreadful distemper
to their children, merely to prevent an uncertain evil;
The English, on their side, call the rest of the Europeans
cowardly and unnatural; Cowardly, because they are afraid
of giving their children a little pain;
unnatural, because they expose them to the risk
of eventually dying of smallpox
(From Voltaire's Letters concerning the English nation, 1733)
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 2
4
Jenner giving
the first vaccination
5
Serum
Infection Infection
Serum from of an individual who has recovered
from an infection will protect others from that infection
(Behring and Kitasato, 1890)
(Slide courtesy J. Sale)
Blood is indeed a very special substance
6
• Induction of specific antibody
• Memory –
maintenance of specific antibody over long periods
• Diversity of antibodies
• Discrimination of self from foreign
• How do antibodies neutralise the infection?
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 3
7
Side-chain theory (Ehrlich, 1900)
8
Figure to illustrate the clonal selection theory of immunity; Contact of the corresponding antigenic determinant Ag;
C with cells of clone cstimulates proliferation to antibody-producing
plasma cells cp and non-antibody producing types c
Clonal selection theory
(Burnet, 1957)
9
The structure of antibodies
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 4
10
Variable region
(millions)
Constant region
(few - IgM, IgG, IgA, IgE)
Antigen
Biological effectors
11Immunobilogy, 6/e. (© Garland Science 2005)
12Immunobilogy, 6/e. (© Garland Science 2005)
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 5
13
Ag-binding site formed
by loops at top of β-strands
14
15
IgMIgMIgMIgM Pre-immune serum Ab
IgGIgGIgGIgG Major immune serum Ab
(IgG1, 2, 3, 4 subclasses in man)
(IgG1, 2a, 2b, 3 subclasses in mouse)
IgAIgAIgAIgA Ab in secretions (colostrum; milk; gut)
(IgA1, 2 subclasses in man)
IgEIgEIgEIgE Anti-parasite/allergy (low amounts)
IgDIgDIgDIgD Surface receptor
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 6
16
How is the diverse repertoire of antibody
specificites generated?
17
Clonal selection theory
(Burnet, 1957)
Figure to illustrate the clonal selection theory of immunity; Contact of the corresponding antigenic determinant Ag;
C with cells of clone cstimulates proliferation to antibody-producing
plasma cells cp and non-antibody producing types c
18
Germlinetheory
Somatic mutation theory
Conventional
cell-specific
expresssion
Generator
of
diversity
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 7
19
Variable region
(millions))))
Constant region
(few - IgM, IgG, IgA, IgE)
Antigen
Biological effectors
20
Gene rearrangement
Somatic hypermutation
Cleav
e
Gene conversion
21
A repertoire of functional antibodies is generated by gene rearrangement
(V(D)J joining)
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 8
22Immunobilogy, 6/e. (© Garland Science 2005)
23Immunobilogy, 6/e. (© Garland Science 2005)
24Immunobilogy, 6/e. (© Garland Science 2005)
Figure 4-3
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 9
25Immunobilogy, 6/e. (© Garland Science 2005)
26
RSS st ructures:RSS st ructures:RSS st ructures:RSS st ructures:
C odingC odingC odingC oding N oncodingN oncodingN oncodingN oncoding12 12 12 12 BPBPBPBPSpacerSpacerSpacerSpacer
C odingC odingC odingC oding N oncodingN oncodingN oncodingN oncoding23 23 23 23 BPBPBPBPSpacerSpacerSpacerSpacer
CA CAGTGCA CAGTGCA CAGTGCA CAGTGG T GTCACG T GTCACG T GTCACG T GTCAC
CA CAGTGCA CAGTGCA CAGTGCA CAGTGG T GTCACG T GTCACG T GTCACG T GTCAC
A CA AA AACCA CA AA AACCA CA AA AACCA CA AA AACCT G TTTTTGGT G TTTTTGGT G TTTTTGGT G TTTTTGG
A CA AA AACCA CA AA AACCA CA AA AACCA CA AA AACCT G TTTTTGGT G TTTTTGGT G TTTTTGGT G TTTTTGG
(e.g., the ‘V’)
(e.g., the ‘J’)
• The RAG (Rearrangement Activating Gene)
recombinase catalyses V(D)J joining
• RAG recognises RSSs (Rearrangement Signal Sequences)
27
V(D)J recombination is restricted
by the 12/23 rule
23232323
VVVV12121212
JJJJ
Integrated VJ
VVVVJJJJ
Spacer DNA containing
the signal sequences
2323232312121212
9999----mermermermer7777----mermermermer
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 10
28
Ordered rearrangement
of the IgH loci
during development
2 32 32 32 3 2 32 32 32 31 21 21 21 2 1 21 21 21 2
VVVV HHHHDDDD HHHH JJJJ HHHH
2 32 32 32 3
JJJJ HHHH
IgH locus
29
RAG locus
RAG-1 RAG-2
30
Known NHEJ factors:
KUKUKUKU7 07 07 07 0, KU, KU, KU, KU80808080, DNA, DNA, DNA, DNA----PKcs, PKcs, PKcs, PKcs, A rtemis, XRCCArtemis, XRCCArtemis, XRCCArtemis, XRCC4444, ligase , ligase , ligase , ligase 4444
NHEJ =
Non-Homologous
End-Joining
NHEJ needed to repair
double-strand DNA breaks-
however caused (e.g., by X-rays)
NHEJ
DSBsSi gnalSi gnalSi gnalSi gnalC odingC odingC odingC oding
S ignalS ignalS ignalS ignal
CodingCodingCodingCoding
VVVV JJJJ VVVV JJJJ
R SSsR SSsR SSsR SSs
R SSsR SSsR SSsR SSsVVVV JJJJ
V(D)J recombination
Precise RS
and modified coding joins
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 11
31
Initiation of V(D)J recombination
5 '5 '5 '5 '3333 ''''
OHOHOHOH
PPPP5555 ''''3333 ''''
R AGR AGR AGR AG1111/RAG/RAG/RAG/RAG2222
V
V
RSS
RSS
V
N uc leophilic attackN uc leophilic attackN uc leophilic attackN uc leophilic attack
OHOHOHOH PPPP5 '5 '5 '5 '3333 '''' RSS
3333 ''''
OHOHOHOH PPPP5 '5 '5 '5 ' V RSS
32
TdT evolved to diversify
antigen receptor repertoires
via junctional diversification
Terminal deoxynucleotidyl transferase (TdT) -
a lymphocyte-specific enzyme
that adds nucleotides to DNA 3’-ends
in a non-templated manner
33
Junctional sequences
of IgH rearrangements
Germline TGTGCAAGACA
22A2-4 TGTGCAAGA
22A2-10 TGTGCAAGA
22A2-13 TGTGCAAGAC
22A2-3 TGTGCAAGACA T
22A2-11 TGTGCAAGACA T
22B2-4 TGTGCAAG
22B2-1 TGTGCAAGAC
22B2-7 TGTGCAAGACA T
22A2-6 TGTGCAAGACA
22A2-9 TGTGCAAGAC
ACTACTTTGACTACTGG
ACTACTTTGACTACTGG
TACTTTGACTACTGG
TTTGACTACTGG
ACTACTGG
ACTACTTTGACTACTGG
ACTACTTTGACTACTGG
ACTACTTTGACTACTGG
GG
TACTACTTTGACTACTGG
TGACTACTGG
TTTGACTACTGG
GCCCCCA
GG
AG
AG
GC
A
GGG
T
G
CC
CC
GAGG
AAGGGC
TAACTGG
TCATTACGAC
TACTATGATTACGAC
GATTACGAC
AGTATGGTAAC
AGTATGGTAAC
CTATGGTAACT
GGTTACT
CTATGATGGT
ACTATAGGTACGAC
TATAGGTACGAC
GG
G
G
T
T
GT
T
VH81X P N P D P N P JH3TdTTdTTdTTdT + /+ /+ /+ /----
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 12
34
V(D)J recombination
RSS
Coding segment
RAG1/RAG2 bindingRAG1/RAG2 cleavage
Hairpin coding ends
Blunt, 5' phosphorylated
RS endsKu binding to DNA ends
NHEJ factors are required
to join RS and coding ends
DNA-PKcs is involved
in coding end joining
And to a lessor degree
in RS joining
DNA-PKcs/Artemis complex opens coding end hairpins
Opened coding ends
may be processed
by Artemis?Processing of coding
ends by TdT
TdT
Fill-in and ligation of coding ends
by Lig4/XRCC4
Perfect ligation
of RS ends by XRCC4/Lig4
Imprecise/variable coding join
Precise signal join
35Immunobilogy, 6/e. (© Garland Science 2005)
36
A repertoire of functional antibodies
is generated by gene rearrangement
(V(D)J joining)
VDJ C-mu
Diversity
IgM (C µ)
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 13
37
-> Clonal selection theory leads to prediction
that B cells must have a receptor for antigen on their cell surface
Clonal selection theory
(Burnet, 1957)
38
Antibodies exist both in secreted
form and as membrane-integral
receptors for antigen
39Immunobilogy, 6/e. (© Garland Science 2005)
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 14
40Immunobilogy, 6/e. (© Garland Science 2005)
41
Selection
Maturation to yield high affinity antibodiesdepends on somatic mutation
CγγγγCµµµµ
µµµµ Cγγγγ
γγγγ
42
Developing from an IgM to an IgG responsedepends on class switch recombination
Primary IgM repertoire
Affinity maturation
Class switching
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 15
43
Somatic hypermutation
44
Somatic mutation
of immunoglobulin genes
Preferred target: AGCT
Transition preferred
over transversion
The variable region
of an immunoglobulin gene
is targeted for somatic
mutation in B lymphocytes
MutatorMutator is recruited by transcription
regulatory elements
45
Somatic hypermutation
Gene conversion
Gene rearrangement
Cleav
e
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 16
46
Immunoglobulin gene conversion
47
Birds and rabbits use gene conversion
48
Developing from an IgM to an IgG responsedepends on class switch recombination
Primary IgM repertoire
Affinity maturation
Class switching
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 17
49Immunobilogy, 6/e. (© Garland Science 2005)
50Immunobilogy, 6/e. (© Garland Science 2005)
51
Assembled by gene rearrangement
-----------------------------------------------------
Diversified by:
Variable region (antigen-binding)
Somatic hypermutation
Gene conversion
Constant region (effector activity)
Class-switch recombination
Functional antibody genes
Recombination-activation
genes - RAG1/2
All dependent
upon AID
(activation-
induced
deaminase)
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 18
52
AID was discovered as a B cell-specific gene
which, when inactivated, leads to deficiency in
• Somatic mutation,
• Class switching and
• Gene conversion
The sequence of AID shows similarity
to that of bacterial enzymes
which deaminate dCTP->dUTP
53
So, three distinct processes:
• Switch recombination
• Somatic hypermutation
• Ig gene conversion
are all triggered by a single enzyme (AID)
How?
54
Somatic
mutation
Gene conversion
Switch
recombination
AIDdeamination
Cytosine Uracil
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
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55
Errors during copyingMismatch
repair
Environmental damage
X-rays; uv light
Chemicals
(Oxidation; alkylation..)
Spontaneous breakdown
Excision
repair
End-
joining
Recombinational
repair
56
57Courtesy John Tainer and Laurence Pearl
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 20
58
1. During somatic hypermutations:
• Replication over the UG lesion
• During subsequent divisions the U will get replaced by T
• Giving rise to transition mutation
2. Replication over abasic site
• The polymerase will hold overthe abasic site
• Essentially any base can get inside
• Giving rise to either transitionor transvertion
3. Recognition by the MSH2/MSH6 mismatch complex
• Initiation of a form of DNA patch
repair mechanism
• Leads to mutagenic patch repair over the AT lesion
• During gene conversion,
the abasic site generated
by uracil excision,
acts as a trigger
for recombinational repair
• During switch recombination
• The nick probably made
by AP-endonuclease,
is presumably converted
to a double strand break
• Cγ or Cα is brought in place of Cµ
59
Evidence for DNA deamination mechanism:
• AID is capable of mutating DNA
by deaminating C -> U in DNA
60
Vector AID
Wild Type
Deficient
in uracil-DNA
glycosylase
repair enzyme
AID can mutate bacteria
Screen for frequency of rifampicin-resistant mutants
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
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61
AID can deaminate a single-stranded
DNA oligonucleotide in vitro
62
Evidence for DNA deamination mechanism:
• Perturb antibody diversification pathways
through deficiency in proteins
that recognise and process U:G lesions
63
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
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64
Simultaneous deficiency in both UNG and MSH2
completely blocks Ig class switching
65
66
L e sion bypassL e sion bypassL e sion bypassL e sion bypass
Re combinationRe combinationRe combinationRe combination----mediated mediated mediated mediated re p airre p airre p airre p air
B ase B ase B ase B ase e x cisione x cisione x cisione x cision
E n dE n dE n dE n d----joiningjoiningjoiningjoining
Somatic
mutation
Gene conversion
Switch
recombination
AIDdeamination
Cytosine Uracil
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 23
67Immunobilogy, 6/e. (© Garland Science 2005)
Many lymphoid cancers
are associated with chromosomal
translocations caused
by aberrant V(D)J joining (RAG)
or aberrant Ig class switching (AID);
Follicular B cell tumours
also often have oncogene point mutations
that look as if they are caused by AID
Mistargeted gene rearrangements
and mutation can cause cancer
68
Anal agous to transposases Anal agous to transposases Anal agous to transposases Anal agous to transposases and viral integrasesand viral integrasesand viral integrasesand viral integrases
????
69
AID’s ancestors worked on free
bases/nucleos(t)ides
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
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70
Are there other AID-related proteins
which deaminate C->U
in polynucleotides?
71
HAE PC..C
dCMP DA, cytidine DA
HAE PC..C
AID
HAE PC..C
APOBEC3
F.F.F F ITWF SWS ARLY FVE ILLL.L.L L.L.L
HAE PC..C
APOBEC1
HAE PC..C
APOBEC2
72
dCMP DA, cytidine DA
Free CNucleotide
metabolism
AID C in Ig DNAIg Gene
Diversifictn
APOBEC1 C in ApoB RNAEditing
ApoB mRNA
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 25
73
APOBEC1 catalyses C->U deamination in the RNA transcript for apolipoprotein B
(a fat emulsifying protein)
DNA
Protein
RNA
ApoBApoBApoBApoB----long (liver)long (liver)long (liver)long (liver) ApoBApoBApoBApoB----short (intestine)short (intestine)short (intestine)short (intestine)
APOBEC1
74
APOBEC2 is as old as AID (all vertebrates) and performs an unknown function
in muscle
75
AID APOBEC2
AID APOBEC2Cartilaginous/bony fish
Birds, reptiles
Cytidine/dCMPdeaminases
Vertebrates
APOBEC1AID APOBEC2Marsupials
APOBEC1AID APOBEC2Placentals APOBEC3
A n tiA n tiA n tiA n ti ----viralsviralsviralsvirals
APOBEC1AID APOBEC2Primates APOBEC3A-HM u scle;M u scle;M u scle;M u scle;
f u nction?f u nction?f u nction?f u nction?I g geneI g geneI g geneI g gene
d i versifictnd i versifictnd i versifictnd i versifictnA p oB mRNAA p oB mRNAA p oB mRNAA p oB mRNA
e d itinge d itinge d itinge d iting
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 26
76
Adaptive
immunity
Innate
immunity
Cytosine Uracil
AID - a deaminase
which targets
antibody genes
APOBEC3G -
a deaminase
which targets
viruses (HIV)
77
Introduce an extra copy of an endogenous gene into neurospora
It becomes silenced by:
Methylation at dC residues
Introduction of mutations (RIP):
These are very largely point mutations at dC:dG
So is Neurospora RIP, an ancient antecedent of phase 1
of vertebrate antibody somatic mutation?
Not quite
RIP - repeat-induced point mutation
78
dC deaminationdC methylation
The Generation of Diversity
in Antibody Genes
Prof. Michael Neuberger
The screen versions of these slides have full details of copyright and acknowledgements 27
79
80