The Generation of Diversity in Antibody Genes Prof. Michael … · in Antibody Genes Prof. Michael...

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


in Antibody Genes


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)


in Antibody Genes



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?


in Antibody Genes



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


in Antibody Genes



10

Variable region

(millions)

Constant region

(few - IgM, IgG, IgA, IgE)

Antigen

Biological effectors

11Immunobilogy, 6/e. (© Garland Science 2005)



in Antibody Genes



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


in Antibody Genes



16

How is the diverse repertoire of antibody

specificites generated?

17


(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


in Antibody Genes



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)


in Antibody Genes






Figure 4-3


in Antibody Genes




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


in Antibody Genes



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


in Antibody Genes



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 + /+ /+ /+ /----


in Antibody Genes



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


36

A repertoire of functional antibodies

is generated by gene rearrangement

(V(D)J joining)

VDJ C-mu

Diversity

IgM (C µ)


in Antibody Genes



37

-> Clonal selection theory leads to prediction

that B cells must have a receptor for antigen on their cell surface


(Burnet, 1957)

38

Antibodies exist both in secreted

form and as membrane-integral

receptors for antigen



in Antibody Genes




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


in Antibody Genes



43


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


Gene conversion

Gene rearrangement

Cleav

e


in Antibody Genes



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


in Antibody Genes





51

Assembled by gene rearrangement

-----------------------------------------------------

Diversified by:

Variable region (antigen-binding)


Gene conversion

Constant region (effector activity)

Class-switch recombination

Functional antibody genes

Recombination-activation

genes - RAG1/2

All dependent

upon AID

(activation-

induced

deaminase)


in Antibody Genes



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


in Antibody Genes



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


in Antibody Genes



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


in Antibody Genes



61

AID can deaminate a single-stranded

DNA oligonucleotide in vitro

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Evidence for DNA deamination mechanism:

• Perturb antibody diversification pathways

through deficiency in proteins

that recognise and process U:G lesions

63


in Antibody Genes



64

Simultaneous deficiency in both UNG and MSH2

completely blocks Ig class switching

65

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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


in Antibody Genes




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


in Antibody Genes



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


in Antibody Genes



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

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APOBEC2 is as old as AID (all vertebrates) and performs an unknown function

in muscle

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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


in Antibody Genes



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Adaptive

immunity

Innate

immunity

Cytosine Uracil

AID - a deaminase

which targets

antibody genes

APOBEC3G -

a deaminase

which targets

viruses (HIV)

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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


in Antibody Genes



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The Generation of Diversity in Antibody Genes Prof. Michael … · in Antibody Genes Prof. Michael...

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