B cell zone

Vertiefungsmodul Immunbiologie
Ringvorlesung
Erlangen  WS13/14
Adaptive Humorale Immunität
Etablierung des sekundären Antikörper-Repertoires
Hans-Martin Jäck
Abteilung für Molekulare Immunologie
Medizinische Klinik III
Nikolaus-Fiebiger-Zentrum
FAU Erlangen-Nürnberg
Plasma Cell Differentiation
2
Memory
B cell
Naive
B cells
Ag + TH
Germinal Center
Reaction
&
AID
More affine &
specialized
antibodies
„Memory“
Plasma cell
THEMEN
 Überblick: Adaptive humorale Immunität
 Keimzentrumsreaktion, Affinitätsreifung und
IgH-Klassenwechsel
 AID und APOPECs
 Funktion und Wirkmechanismus von AID
 AID und angeborene Immunität
Division of Molecular Immunology, Universitätsklinikum Erlangen
3
Immunsystem bildet Barrieren
Angeboren
 Physikalisch
•
•
•
•
Haut
Schleim
Darmflora
Flimmerhaare
Keime & fremde
Substanzen
•
•
•
•
•
Bakterien
Pilze
Würmer
Viren
Fremde Eiweiße
 Physiologisch
• pH
• Temperatur
 Zellulär (Leukozyten)
• Makrophagen
• Granulozyten
• Lymphoyzen
 Entzündung
• Die vier ‚ors‘
BARRIEREN
Abwehrsystem =
IMMUNSYSTEN
Erworben
 B- und T-Zellen
 Antikörper
 T-Zell-Botenstoffe
•
•
•
•
Interleukine
Zytokine
Lymphotoxine
Chemokine
 Gedächtnis !!!!
 Adaptive Immunität
(Immunitas, lat.: ‚Freisein
von Leistungen/Lasten‘)
Division of Molecular Immunology, Universitätsklinikum Erlangen
4
Adaptive humorale
Immunität
Anatomie der Adaptiven Immunität
Lymphknoten
T-Zellzone (extrafollikulär)
DZ
MHC II
MHC I
Naive
CD8-T
TK
MHC II
Naive
CD4-T
Dendritische
Zelle (DZ)
B-Zellzone (follikulär)
Gedächtnis-B
B
Infektion
TH
Division of Molecular Immunology, Universitätsklinikum Erlangen
TFH
Plasmazelle
6
Antibodies – Structure and Function
 Glykoproteine
Antigenbindung
 Quartärstruktur
VH
• 2 identische schwere (H) (ca. 50kDa)
• 2 identische leichten (L) Ketten (ca. 25kDa)
• Verknüpft über Inter-Ketten-S-S-Brücken (
CH1
VL
L
CL
CH2
Effektorfunktion
CH3
CHO
 Ketten enthalten variable (V) und
konstante (C) Regionen
H
 Ketten bestehen aus Ig-Domänen
• Stabilisiert über Intraketten-S-S-Brücken (
)
)
 Diversität der V- und C-Regionen
IgM
• Milliarden verschiedener V-Regionen (Idiotyp)
• 2 CL: k und l; 5 CH: m, d, g, a und e (Isotyp)
Division of Molecular Immunology, Universitätsklinikum Erlangen
7
Antibodies – Structure and Function
Antigen binding sites
= Paratop
“Magic Part”
3 loops (fingers) from each V region
form the antigen binding site (paratop)
CDRs = complementary determining regions 1-3
VH
VH
VL
VL
1
CH
CL
3
L
CH
Effector
sites
“Bullet Part”
3
2
CH
H
CH





2
1
Tissue distribution
Serum halflife
Complement
Phagocytosis
Recruting of cells
1
2 3
Janeway
Antibodies are bifunctional (Paul Ehrlich‘s Magic Bullets)
Division of Molecular Immunology, Universitätsklinikum Erlangen
8
Antibodies – Effector Functions

Neutralisation

Agglutination

Activation of Complement

Enhancement of Phagocytosis
(Opsoniation)

Recrutement of effector cells
(Neutrophils, natural killer cells)
Division of Molecular Immunology, Universitätsklinikum Erlangen
9
Adaptive Humoral Immunity
Generation of the Primary
B Cell Repertoire
B cell
receptor
1
Antigen
1
One B cell - One Receptor
Maturation of B Cells
Central Maturation
(Bone marrow)
BCR
Pre-BCR
Stem
cell
Late
Pro-B
Early
Pre-B
VH→D → JH
Late
Pre-B
Immature
B cell
VL → JL
L
H
Division of Molecular Immunology, Universitätsklinikum Erlangen
11
Generation of Antibody Diversity
S. Tonegawa
Nobel Price 1987
Basel Institute
of Immunology
ca. 2.5 Mb (mouse)
D segments
V segments
HC locus
J segments
Cm
stem cell
Recombination
J
V
B cell
Ck
VH Exon
Transcription
Translation
VH
VL Exon
Cm
C B cell
N
LC locus
N
kL chain
C
µH chain
V(D)J recombination generates antibody diversity
Division of Molecular Immunology, Universitätsklinikum Erlangen
12
Summary: Preimmune Repertoire
134 VH
13 D
4JH
Cm
Recombinatorial diversity
• Random assembly from V, D & J
Combinatorial diversity
ca. 107
antibodies
109-1012
antibodies
• Random pairing of H & L chains
Junctional diversity
• Unprecise V(D)J joining
• Nucleotide (N) addition (TdT)
• Usage of three RF in D segments
B-Zellen
Anzahl
Neu/Tag
Division of Molecular Immunology, Universitätsklinikum Erlangen
Mensch
1012
109
Maus
109
106
13
HUMORAL IMMUNE RESPONSE
Central Maturation
Peripheral Maturation
(Bone marrow)
(Spleen)
BCR
Pre-BCR
Stem
cell
Late
Pro-B
Early
Pre-B
VH→D→ JH
Late
Pre-B
Immature
B cell
Transitional
B cells
Mature
B cell
VL → JL
Primary
repertoire
L
H
~109-1012 specificies
Division of Molecular Immunology, Universitätsklinikum Erlangen
14
HUMORAL IMMUNE RESPONSE
15
Central Maturation
Peripheral Maturation
Effector Phase
(Bone marrow)
(Spleen)
(lymph node, spleen, etc.)
Plasma
cell
BCR
Pre-BCR
Ag +
TH
Stem
cell
Late
Pro-B
Early
Pre-B
VH→D→ JH
Late
Pre-B
Immature
B cell
Transitional
B cells
Mature
B cell
H
Memory
B cell
VL → JL
Primary
repertoire
L
GC
~109-1012 specificies
Division of Molecular Immunology, Universitätsklinikum Erlangen
Secondary
repertoire
→ Affnity maturation
→ Effector functions
Adaptive Humoral Immunity
Generation of Effector B Cells
IgM
Plasma
cell
Ag + TH
Ag + TH
IgD
Memory B
IgG
Anatomie der Adaptiven Immunität
Lymphknoten
T-Zellzone (extrafollikulär)
DZ
MHC II
MHC I
Naive
CD8-T
TK
MHC II
Naive
CD4-T
Dendritische
Zelle (DZ)
B-Zellzone (follikulär)
Gedächtnis-B
B
Infektion
TH
Division of Molecular Immunology, Universitätsklinikum Erlangen
TFH
Plasmazelle
17
Activation of Naive CD4+ T Cells
King et al., Annu. Rev.
Immunol. 2008
cytokines
Dendritic cell
Activation of
naive
CD4+ T cells
in T cell zone
Division of Molecular Immunology, Universitätsklinikum Erlangen
B cell help
in follicule
18
B Cell Antigens
The World of Antigens
(Antibody generating)
Differentiation
Clonal Expansion
IgM
Ig receptors recognize
• Proteins
• Lipids
• Nuclei acids
Ag
IgM
• Carbohydrates
• Organich molecules or
Haptens (Half-Ag)
• Metals
• Plastic
+/-TH
+/-TH
IgD
Naive
B cells
But only proteins are good T
cell-dependent antigens
Division of Molecular Immunology, Universitätsklinikum Erlangen
Short-lived
Plasma cells
IgG, IgA, IgE
+TH
Long-lived
plasma cells
+TH
Memory
B cell
19
T Cell Antigens
T-Zellrezepror erkennt Fremd (Peptid) und Selbst (MHC)
(MHC restiction - Zinkernagel & Doherty )
Ag
B-ZellRezeptor
(BZR)
T-ZellRezeptor
(TZR)
Dendritische
Zelle
MHC II
MHC I
Ag-Prozessierung
& Präsentation
Division of Molecular Immunology, Universitätsklinikum Erlangen
20
Merkmale der adaptiven Immunität
Organimsus erinnert sich an Antigen und antwortet mit einer
 besseren
(über Affinitätserhöhung)
 spezialisierteren
(durch IgH-Klassenwechsel)
 schnelleren
(Signalwege ?)
auf das jeweilge Pathogen zurechtgeschnitte Antikörperantwort
Division of Molecular Immunology, Universitätsklinikum Erlangen
21
Anatomical Location
Secondary
lymphatic
organs
B cell
zone
Primary
B cell
follicule
Lymph
nodes
medulla
arteria
Appendix
Paracortex
(T cell zone)
venule
Spleen
Tonsils
Peyer
Plaques
Afferent
lymphatic
vessel
Secundary
B cell follicle
„Germinal center“
Efferent
lymphatic
vessel
From Janeway
Division of Molecular Immunology, Universitätsklinikum Erlangen
22
Anatomy of B Cell Response
Spleen section - 7 days SRBC
B
T Cell Zone
T
HEV
Antigen
Expansion
IgM
IgM
+/-TH
Naive
B cell
+TH
B cell
focus
GC
B Cell Zone
CXCR5
IgD
PNA
CD3
- B cells
- GC B cells
- T cells
Short-lived
Plasma cell
IgG
IgA
IgE
Memory
B cell
Primary
follicle
IgG
IgA
IgE
Secondary
follicle
+
Germinal center
Long-lived
plasma cells
T-Zell-abhängige B-Zellaktivierung
Lymphknoten
T-Zellzone (extrafollikulär)
DZ
MHC II
MHC I
Naive
CD8-T
TK
MHC II
Naive
CD4-T
Dendritische
Zelle (DZ)
B-Zellzone (follikulär)
Gedächtnis-B
B
Infektion
TH
Division of Molecular Immunology, Universitätsklinikum Erlangen
TFH
Plasmazelle
24
Anatomy of B Cell Response
Spleen section - 7 days SRBC
B
T Cell Zone
T
HEV
Antigen
B Cell Zone
- B cells
- GC B cells
- T cells
IgM
IgM
+TH
Naive
B cell
GC
IgD
PNA
CD3
Expansion
Primary
follicle
B cell
focus
Short-lived
Plasma cell
Extrafollikuläre B/T-Zell-Kooperation
OH
NO2
Träger
B-Zell-Epitop
(Peptid oder
Hapten)
IL2/4/5
Träger
T-Zell-Epitop
(Peptid)
Gekoppelte Erkennung
(Linked recognition)
B- und T-Zellepitop müssen
auf dem gleichen Molekül
liegen
ILR
CD40
1
2
CD40L
B
+
Primed TH
TH
MHC II
+
TZR
Peptid
Immunologische
Synapse
BZR
CD40L-Defizienz
• Keine Antikörper gegen
Proteine (z.B. Tetanus)
• Kein Klassenwechsel
• Kein Gedächtnis
→ Keine Schutzimpfung
• Aber gute Antwort gegen
Kohlenhydrate !!!!
→ Viel IgM im Serum
→ Hyper-IgM-Syndrom I
Division of Molecular Immunology, Universitätsklinikum Erlangen
26
STUDON
RINGVORLESUNG
http://www.studon.uni-erlangen.de/crs816430.html
Anatomy of B Cell Response
Spleen section - 7 days SRBC
B
T Cell Zone
T
HEV
Antigen
Expansion
IgM
IgM
+/-TH
Naive
B cell
+TH
B cell
focus
GC
B Cell Zone
CXCR5
IgD
PNA
CD3
- B cells
- GC B cells
- T cells
Short-lived
Plasma cell
IgG
IgA
IgE
Memory
B cell
Primary
follicle
IgG
IgA
IgE
Secondary
follicle
+
Germinal center
Long-lived
plasma cells
Germinal Center (dt.: Keimzentrum)
1884
Fleming discovers germinal centers. The name ‘GC’
is based on Fleming’s finding that GC contain a high
mitotic activity. He believed that GC are the site of
germination or lymphopoiesis
1920
The idea that GC are site of lymphopoiesis fell short
because it did not fit the transient appearance
1924
Latta and West proposed that GC are rather sites of
death and senescence that lymphopoiesis
1940-43
Crabb and Kelsall, and Hellman found that the
presence of GC correlates with chronic antigenic
stimulation and that GC can be induced by
immunization
GC
Elise Punkenburg
Bachelorarbeit, Erlangen 2008
As we know now, GC are the site of local proliferation and cell death, both of which
contribute in antibody affinity maturation and formation of memory cells
Division of Molecular Immunology, Universitätsklinikum Erlangen
29
Germinal Center Reaction
Germinal
Center
FDC
Light
zone
30
Modified from
McHyzer-Williams
2011
B cell
zone
TFH
IgG, IgA, IgE
Memory
B cell
Selection
?
GC exit
Dark
zone
?
“Memory”
plasma cell
(long-lived)
Expansion
SHM
IgG, IgA, IgE
CSR?
CSR
IgM
TFH
T cell
zone
GC - Molecular Changes at the Ig locus
IgM
IgG, IgA, IgE
VH
AID
VL
CL
31
CH
1. Somatic hypermutation
2. IgH class switch
Better and more specialized antibodies
IgH Class Switch Recombination (CSR)
Kinoshita & Honjo NRCB(2001)
Division of Molecular Immunology, Universitätsklinikum Erlangen
32
IgH Class Switch Recombination (CSR)
DNA-Looping-out und Deletion
VH
Cm
IL4
LPS
Cd
Cg3
Sm
IgM
Sg3
CSR
VH
•
•
•
•
IgG1
Sg1
Cg2b Cg2a
Sg2b
Cg1
Sg2a
Ce
Se
Ca
Sa
S, switch regions
Cytokine
Cg3
Cm
Cd
Cg1
AID
Cg2b Cg2a
Ce
Ca
Cg2b Cg2a
Ce
Ca
Synapsis
Incision
Double-strand breaks
End-Joining/Ligation
VH
Cg1
Switch circle
Jäck et al.,P.N.A.S. USA 1988
von Schwedler et al., Nature 1990
Division of Molecular Immunology, Universitätsklinikum Erlangen
33
Somatic Hypermutation (SHM) of V Regions
SHM und Ig-Mutator
Jacob et al., Nature 1991
V
AID
Einfügen von Punktmutationen willkürlich
über das gesamte V-Exon des L- und HKetten gens verteilt
Division of Molecular Immunology, Universitätsklinikum Erlangen
34
Germinal Center Reaction - Selection
Follicular
dendritc cell
(FDC)
FcR
Selection
CR
C
Light
zone
native
antigen
B cell
with high-affine
Ag receptor
Dark
Zone
35
Affinity Maturation
STEP 1:
Somatic hypermutation over
entire V exons
STEP 2:
FDC selects B cells with higher
affinity for immunizing antigen
Ag-specific antibodies
with higher affinity
PROBLEM:
Self-reactive B cells could be selected by self-antigen on FDC –
► Requirement for another checkpoint
Division of Molecular Immunology, Universitätsklinikum Erlangen
36
Germinal Center Reaction
FDC
Selection
TFH
Selected
B cell
Dark
zone
Expansion
SHM
AID
Modified from
McHyzer-Williams
2011
B cell
zone
GC reaction
Light
zone
37
CSR?
CSR
IgM
TFH
T cell
zone
B/T-Kooperation im Keimzentrum
Nutt & Tarlinton, Nat Immunol. 2011
Division of Molecular Immunology, Universitätsklinikum Erlangen
38
Germinal Center Reaction
FDC
Modified from
McHyzer-Williams
2011
B cell
zone
GC reaction
Light
zone
39
TFH
IgG, IgA, IgE
Memory
B cell
Selection
Selected
B cell
Dark
zone
?
GC exit
IgG, IgA, IgE
“Memory”
plasma cell
(long-lived)
Expansion
SHM
AID
?
CSR?
CSR
IgM
TFH
T cell
zone
Control of PC Differentiation
Staudt/
Calame/
Lassila
Model
PC Program
GC Program
Pax5
AID
Repair
40
Xbp1
IRF4 ↑
Bcl6
Blimp1
Bach2
IRF4↑↑↑↑
MiTF
Modified from Nutt et al., 2011
Germinal Center Reaction
FDC
Modified from
McHyzer-Williams
2011
B cell
zone
GC reaction
Light
zone
41
TFH
IgG, IgA, IgE
Memory
B cell
Selection
?
GC exit
Dark
zone
IgG, IgA, IgE
“Memory”
plasma cell
(long-lived)
Expansion
SHM
AID
?
CSR?
CSR
IgM
TFH
T cell
zone
Effector B Cells
IgA
IgG, IgE
Plasma Cell (PC)
• Igmem → Igsec
• Ig production ↑ (100x)
• Long-lived PC in bone marrow
niches
IgM
IgM
Ag
Plasma
cell
Memory B Cell
IgD
IgG
IgA
IgE
Naive
B cell
Memory
B cell
•
•
•
•
IgGmem, IgAmem or IgEmem
Reacts faster to Ag
Circulates through body
Long-lived (does not require
antigen contact for survival)
• Generation requires T help
Division of Molecular Immunology, Universitätsklinikum Erlangen
42
Summary: Effector B Cells
Differentiation
Clonal Expansion
IgM
Better and more
IgM
Ag
IgM
+/-TH
+/-TH
IgD
Naive
B cells
Short-lived
Plasma cells
specialized abs
IgG, IgA, IgE
Germinal center
reaction
+TFH
IgG, IgA, IgE
• Proliferation
Long-lived
plasma cells
• Somatic hypermutation
+TFH
• Selection
• class switch
• Effector cells
AID
Division of Molecular Immunology, Universitätsklinikum Erlangen
Memory
B cell
43
Adaptive Humoral Immunity
AID
Activation-Induced Deaminase
Master regulator of secondary
antibody diversification
and ?????
AID - Entdeckung
 Entdeckt über substraktive Hybrisierung als induzierbares Gen in einer
B-Lymphomlinie (CH12) mit IL4-induzierbarem IgH-Klassenwechsel
(Muramatsu et al. JBC 1999)
 Synthese induziert in Ag-aktivierten Keimzentren-B-Zellen
 Konvertiert in ssDNA ein C zu einem U
(oxidative Deaminierung)
Deamidase
H2O
Cytosin
AID
NH3
O
Uracil
R C
NH2
Säureamid
O
R C
OH
Carbonsäure
→ Activation-Induced Deaminase = AID (Gensymbol AICD)
 Notwenig für CSR und SHM
Division of Molecular Immunology, Universitätsklinikum Erlangen
45
Kurzer Ausflug in APOBEC-Familie
Mensch
 Enthalten alle Deaminase-Domäne
mit konservierter katalytischer
Stelle (rot)
 Maus besitzt APOBEC1, 2, 3 (eine
Form) und AID
 APOBEC1 und APOBEC3 nur in
Säugern
 AID und APOBEC2 in allen
höheren Vertebraten
 Funktionen:
Goila-Gaul and
Strebel,
Retrovirology
5:51, 2008
→ Editieren von RNA und DNA
→ Immunität gegen Viren
→ Inaktivierung von Retroelementen
Division of Molecular Immunology, Universitätsklinikum Erlangen
46
APOBEC1 – Prototyp eines RNA-editierenden Enzyms
Apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1
N
C
APOB100
Leber
AUG
CAA
APOBEC1
(one !!!! mutation in RNA)
AUG
N
UAG
APOB mRNA
UAG
Mut APOB mRNA
Duodenum
UAA
C
APOB48
Division of Molecular Immunology, Universitätsklinikum Erlangen
•
Navaratnam et al., LBC 1993
•
Teng et al., Science, 1993
47
APOBEC3G – Innate Abwehr gegen Viren und Transposon
 Nur in Säugern (Lymphozyten)
 Mutiert neuen ss-cDNA-Strang
RT
APO3
X XX XX
RNA (viral oder
Retrotransposons)
mutierte
cDNA
 Modelle antiviraler Wirkmechanismen von APOBEG3:
(1) Reduziert Bildung viraler Transkripte
Sheehy et al.
Nature 2002
(2) Inaktivierende Mutationen in Virions
(3) Induziert Abbau mutierter Trankripte
(4) Induziert Abau nicht-mutierter Transkripte durch Rekrutierung zellulärer
Nukleasen →Editing-unabh.Mechanismus
Division of Molecular Immunology, Universitätsklinikum Erlangen
48
Kurzer Ausflug: Retroelements
 Endogene Retroelemente (bis zu 45% des menschlichen Genoms)
 Mutatoren, werden aber auch selber mutiert
From M. Wabl
insertional mutagenesis
TLR7
RIG I
MDA5
new RNA
new protein
new cDNA
Adaptive
Immunity
 Biologische Aktivität/Funktion
AIM2
TLR9
APOBEC3
Trex1
AID
o Genduplikationen
o Vergrößerung des Genoms
o Insertionsmutagenese
Division of Molecular Immunology, Universitätsklinikum Erlangen
49
AID – A Hypermutator
Cytosin
→
AID
Uracil
Activation-Induced Deaminase (AID)
 Converts C in ssDNA to U (oxydative deamination)
 Expressed in activated germinal center B cells
 Discovered by Honjo et al. (1999)
 Required for SHM and CSR
 Defekt: Hyper-IgM syndrome type II
Division of Molecular Immunology, Universitätsklinikum Erlangen
50
AID: Required for SHM & CSR
 Transfection into B cell lines induces CSR
 Germline-deficient AID mice have no CSR and SHM
(Muramatsu et al., Cell 2000)
 Patients with mutated AID (autosomal) no CSR and
strongly reduced somatic mutation (Revy ; Durandy et al., Cell
2000)
 AID-deficient mice and patients produce large
amounts of IgM antibodies against PROTEINS (!!!!)
→ Hyper-IgM syndrome type 2
 Also required for Ig gene conversion in chicken B cells
(Arakawa et al., Science)
Division of Molecular Immunology, Universitätsklinikum Erlangen
51
AID: How does it work?
 RNA Editing Model (indirect)
AID is an indirect mutator by editing (like APOBEC1) a
mRNA (or miRNA) encoding (or controlling) a switch
recombinase/Ig mutator
 DNA Mutation Model (direct)
AID acts directly on DNA (introducing C-to-U mutations)
in both processes
•
•
Constans, A. Class /Switch Wars 2004. The Scientist18(18):28
Honjo et al. 2004. Immunity 20:659-68
Division of Molecular Immunology, Universitätsklinikum Erlangen
52
RNA-Editing-Hypothese
N
C
Aktiver „Mutator“
(Endonuklease)
AID
AUG
CAA
UAG
„Mutator“encoding
mRNA
AID↑ in Keimzentrums-B-Zelle
AUG
N
UAA
C
UAG
„Mutator“encoding
mRNA
Inaktiver Mutator
Division of Molecular Immunology, Universitätsklinikum Erlangen
53
RNA-Editing-Hypothese
Hinweise
 AID hat sehr starke Sequenzähnlichkeit mit APOBEC1 (RNAEditierung)
 AID ist hauptsächlich im Zytosol lokalisiert
 De-Novo Proteinbiosynthese ist für CSR notwendig
(Gegenargument: CSR-spezifischer Ko-Faktor wird synthetisiert)
 AID komplexiert im Zytosol mit mRNA
 AID mit N51A-Mutation verliert DNA-Deaminase-Aktivität,
induziert nach Transduktion in AID-defizienten B-Zellen noch
CSR
• Honjo und Mitarbeiter, PNAS 2008 (Originalmanuskript)
• Shivarov et al., Philos Trans R Soc Lond B Biol Sci 2009 (Review)
Division of Molecular Immunology, Universitätsklinikum Erlangen
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DNA Model
SHM
V exon
Cm
Cg
C
C
CC
C
C
CC
U
U
UU
U
U
UU
Base excision and
error prone
A
G
A
G
CSR
Base excision and
mismatch repair
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Ausflug – DNA-Reparatur
Three major DNA repairing mechanisms:
Base excision (BER), nucleotide excision (NER) and mismatch repair (MMR).
• http://www.web-books.com/MoBio/Free/Ch7G.htm
• Peterson and Cote, G&D, 2004
Division of Molecular Immunology, Universitätsklinikum Erlangen
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DNA-Reparatur – Base excison Repair (BER)
• BER repairs damaged DNA throughout
the cell cycle by first removing the
wrong base
• Works only if DNA's bases are modified
by deamination or alkylation.
• Position of the modified (damaged)
base is called the "abasic site" or "AP
site".
• DNA glycosylases (e.g., Uracil-Nglycosidase = UNG) recognize AP site
and remove its base.
• AP endonuclease removes the AP site
and neighboring nucleotides.
• Gap is filled by DNA polymerase I and
DNA ligase.
Division of Molecular Immunology, Universitätsklinikum Erlangen
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DNA-Reparatur – Nucleotide excison Repair (NER)
• In E. coli, proteins UvrA, UvrB,
and UvrC are removedamaged
nucleotides (e.g., dimer induced
by UV light).
• Gap is filled by DNA polymerase I
and DNA ligase.
• In yeast, the proteins similar to
Uvr's are named RADxx ("RAD"
stands for "radiation"), such as
RAD3, RAD10. etc.
Division of Molecular Immunology, Universitätsklinikum Erlangen
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DNA-Reparatur – Mismatch Repair (MMR)
• Important for mutations unable to be
repaired by BER or NER
E.coli
Dam methylates
template at GATC
GATC
• Mut proteins bind to mismatched bp.
(Eukaroytic homologues are MSH1-5 (mismatch
repair homolog), MLS1 (MutL homolog 1) und PMS
(postmeiotic segregation). Mutations of MSH2,
PMS1 and PMS2 are related to colon cancer.
• Activated Mut H binds to GATC and
cleaves unmethylated strand at GATC.
(In eukaryotes, the mechanism to distinguish the
template strand from the new strand is still unclear)
• Distance between the GATC site and the
mismatch can be up to 1,000 bp.
• Exonucleases remove DNA segment
from cleavage site to mismatch.
• DNA polymerase III fills gap.
Division of Molecular Immunology, Universitätsklinikum Erlangen
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DNA-Mutations-Modell (CSR)
• AID deaminates C to U at
accessible sites in S regions
BER
CSR reduced by 95 %
if BER is deficient!
nicks
• Multiple sites of deamination
and UNG/APE-induced nicks
nicks
• Break processing
(Mismatch repair = MMR)
MMR
• Gaps and DSBs produced
• DNA synthesis up to nick
• Blunt ends at DSBs
• S region ligation
Division of Molecular Immunology, Universitätsklinikum Erlangen
DSB
From: Stavnezer et al., Annu. Rev. Immunol. 2008
Blunt DSB
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DNA-Mutations-Modell - Hinweise
 In vitro, AID mutates ssDNA but not
RNA
 Class switch recombination is inhibited
(95%) and somatic hypermutation is
perturbed in UNG-(BER) deficient mice
Division of Molecular Immunology, Universitätsklinikum Erlangen
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AID – Andere Funktionen?
 LINE-1 ist in AID-defizienten B-Zellen erhöht
(unpublished)
 AID findet sich in großen RNA/Proteinkomplexen
und ko-präzipitiert mit LINE-1 (unpublished)
 AID blockiert Line1-Retrotrans-Position in HEKZellen (MacDuff… Harris. 2009. NAR 37:6854–1867)
 AID-defiziente Patienten haben eine höhere
Anfälligkeit für Autoimmunsymptome und
Leukämien
→ Wie kann man das erklären?
Division of Molecular Immunology, Universitätsklinikum Erlangen
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AID – Zusammenfassung
Activated B cell
nucleus
M. Metzer
Promotion 2010
cytoplasm
ADAPTIVE IMMUNITY
AID targets Ig locus
Cytosine  Uracil
CSR
SHM
 Formation of 2
antibody repertoire
INNATE IMMUNITY ?
Host response
to
viral infection
Inhibition of
retroelement
activity
 Protection from
Autoimmune disease
Proliferative disease
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Übersicht: Adaptive humorale Immunität
Primärantwort
Sekundärantwort
IgM
Kurzlebige
Plasmazelle
(IgM)
Ag+TH
Naive
B-Zelle
Ag+TH
Naive
B-Zelle
Keimzentrumsreaktion
GedächtnisB-Zelle
(IgG, IgA, IgE)
Keimzentrumsreaktion
Log CAK im Serum
IgM
Langlebige
Plasmazelle
(IgG, IgA, IgE)
Langlebige
Plasmazelle
(IgG, IgA, IgE)
Kurzlebige
Plasmazelle
(IgM)
GedächtnisB-Zelle
(IgG, IgA, IgE)
Langlebige
Plasmazelle
(IgG, IgA, IgE)
3
IgG
Schneller
und mehr !!!!
2
1
20Ag
10Ag
IgM
IgG
IgM
t (Tage)
0
4
8
0
4
Division of Molecular Immunology, Universitätsklinikum Erlangen
8
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