hans-martin jäck abteilung für molekulare immunologie medizinische klinik iii...
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Hans-Martin JäckAbteilung für Molekulare ImmunologieMedizinische Klinik IIINikolaus-Fiebiger-ZentrumFAU Erlangen-Nürnberg
Adaptive Humorale ImmunitätEtablierung des sekundären Antikörper-Repertoires
Vertiefungsmodul ImmunbiologieRingvorlesungErlangen WS13/14
2
„Memory“Plasma cell„Memory“
Plasma cell
Naive B cellsNaive B cells
Plasma Cell Differentiation
MemoryB cell
MemoryB cell
Ag + TH
Germinal Center Reaction
&
AID
Germinal Center Reaction
&
AID
More affine &specializedantibodies
More affine &specializedantibodies
Division of Molecular Immunology, Universitätsklinikum Erlangen 3
Überblick: Adaptive humorale Immunität
Keimzentrumsreaktion, Affinitätsreifung und IgH-Klassenwechsel
AID und APOPECs
Funktion und Wirkmechanismus von AID
AID und angeborene Immunität
THEMEN
Division of Molecular Immunology, Universitätsklinikum Erlangen 4
Immunsystem bildet Barrieren
BARRIERENBARRIEREN
Physikalisch• Haut• Schleim• Darmflora• Flimmerhaare
Physiologisch• pH• Temperatur
Zellulär (Leukozyten)
• Makrophagen• Granulozyten• Lymphoyzen
Entzündung• Die vier ‚ors‘
Erworben Angeboren
B- und T-Zellen
Antikörper
T-Zell-Botenstoffe• Interleukine• Zytokine• Lymphotoxine• Chemokine
Gedächtnis !!!!
Adaptive Immunität
Abwehrsystem = IMMUNSYSTEN
(Immunitas, lat.: ‚Freisein von Leistungen/Lasten‘)
• Bakterien• Pilze• Würmer• Viren• Fremde Eiweiße
Keime & fremde Substanzen
Adaptive humorale Immunität
Division of Molecular Immunology, Universitätsklinikum Erlangen 6
Anatomie der Adaptiven Immunität
TH
Lymphknoten
DendritischeZelle (DZ)
NaiveCD4-T
NaiveCD8-T
MHC II MHC IDZ
TK
MHC II
T-Zellzone (extrafollikulär)
B
Infektion
TFH
Plasmazelle
Gedächtnis-B
B-Zellzone (follikulär)
Division of Molecular Immunology, Universitätsklinikum Erlangen 7
L
H
Glykoproteine
Quartärstruktur • 2 identische schwere (H) (ca. 50kDa)• 2 identische leichten (L) Ketten (ca. 25kDa)• Verknüpft über Inter-Ketten-S-S-Brücken ( )
Ketten enthalten variable (V) und konstante (C) Regionen
Ketten bestehen aus Ig-Domänen• Stabilisiert über Intraketten-S-S-Brücken ( )
Diversität der V- und C-Regionen• Milliarden verschiedener V-Regionen (Idiotyp)• 2 CL: und ; 5 CH: und (Isotyp)
IgM
CHO
VL
CL
VH
CH1
CH2
CH3
Antigenbindung
Effektor-funktion
Antibodies – Structure and Function
Division of Molecular Immunology, Universitätsklinikum Erlangen 8
VL
VH
CL
CH
CH
Antigen binding sites= Paratop
“Magic Part”
CH
CH
Effector sites
“Bullet Part”
Tissue distribution Serum halflife Complement Phagocytosis Recruting of cells
3 loops (fingers) from each V region form the antigen binding site (paratop)CDRs = complementary determining regions 1-3
1 2 3
3
1
2
3
Janeway
12
Antibodies – Structure and Function
Antibodies are bifunctional (Paul Ehrlich‘s Magic Bullets)
L
H
VL
VH
Division of Molecular Immunology, Universitätsklinikum Erlangen 9
Antibodies – Effector Functions
Neutralisation
Agglutination
Activation of Complement
Enhancement of Phagocytosis (Opsoniation)
Recrutement of effector cells (Neutrophils, natural killer cells)
Adaptive Humoral Immunity
Generation of the Primary B Cell Repertoire
Antigen 1
B cell receptor
1
One B cell - One ReceptorOne B cell - One Receptor
Division of Molecular Immunology, Universitätsklinikum Erlangen 11
LatePro-B
LatePre-B
Immature B cell
Early Pre-B
Stemcell
Central Maturation(Bone marrow)
VH→D → JH VL → JL
H
L
Pre-BCR BCR
Maturation of B Cells
Division of Molecular Immunology, Universitätsklinikum Erlangen 12
µH chain
N C B cell
B cell
VH C
TranscriptionTranslation
VH Exon
V(D)J recombination generates antibody diversity
Recombination
stem cell
J segments CV segments D segments
ca. 2.5 Mb (mouse) HC locus
L chainN C
VL Exon
J V C
LC locus
Generation of Antibody DiversityS. Tonegawa
Nobel Price 1987Basel Institute of Immunology
Division of Molecular Immunology, Universitätsklinikum Erlangen 13
4JH C134 VH 13 D
Recombinatorial diversity• Random assembly from V, D & J
Combinatorial diversity• Random pairing of H & L chains
ca. 107 anti-
bodies
109-1012 anti-
bodiesJunctional diversity• Unprecise V(D)J joining• Nucleotide (N) addition (TdT)• Usage of three RF in D segments
Summary: Preimmune Repertoire
B-Zellen Mensch Maus
Anzahl 1012 109
Neu/Tag 109 106
14
Division of Molecular Immunology, Universitätsklinikum Erlangen
LatePro-B
LatePre-B
Immature B cell
Early Pre-B
Stemcell
Central Maturation(Bone marrow)
Peripheral Maturation(Spleen)
Transitional B cells
Mature B cell
Pre-BCR BCR
VH→D→ JHVL → JL
H
LPrimary
repertoire
~109-1012 specificies
HUMORAL IMMUNE RESPONSE
15
Division of Molecular Immunology, Universitätsklinikum Erlangen
LatePro-B
LatePre-B
Immature B cell
Early Pre-B
Stemcell
Central Maturation(Bone marrow)
Peripheral Maturation(Spleen)
Transitional B cells
Mature B cell
Pre-BCR BCR
VH→D→ JHVL → JL
H
LPrimary
repertoire
~109-1012 specificies
Secondary repertoire
→ Affnity maturation→ Effector functions
Memory B cell
Plasma cell
Ag + TH
Effector Phase(lymph node, spleen, etc.)
GC
HUMORAL IMMUNE RESPONSE
Adaptive Humoral Immunity
Generation of Effector B Cells
IgG
Memory B
Plasma cell
Ag + TH
IgD
IgMAg + TH
Division of Molecular Immunology, Universitätsklinikum Erlangen 17
Anatomie der Adaptiven Immunität
TH
Lymphknoten
DendritischeZelle (DZ)
NaiveCD4-T
NaiveCD8-T
MHC II MHC IDZ
TK
MHC II
T-Zellzone (extrafollikulär)
B
Infektion
TFH
Plasmazelle
Gedächtnis-B
B-Zellzone (follikulär)
Division of Molecular Immunology, Universitätsklinikum Erlangen 18
Activation of Naive CD4+ T Cells
B cell help in follicule
Activation of naive
CD4+ T cells in T cell zone
cytokines
Dendritic cell
King et al., Annu. Rev. Immunol. 2008
Division of Molecular Immunology, Universitätsklinikum Erlangen 19
Ig receptors recognize• Proteins
• Lipids
• Nuclei acids
• Carbohydrates
• Organich molecules or Haptens (Half-Ag)
• Metals
• Plastic
But only proteins are good T cell-dependent antigens
The World of Antigens (Antibody generating)
Ag
IgM
IgD
NaiveB cells
Short-livedPlasma cells
IgM
+/-TH
DifferentiationClonal Expansion
+/-TH
MemoryB cell
IgG, IgA, IgE
+TH
Long-lived plasma cells
+TH
B Cell Antigens
Division of Molecular Immunology, Universitätsklinikum Erlangen 20
T Cell Antigens
B-Zell-Rezeptor
(BZR)
Ag
T-Zell-Rezeptor
(TZR) MHC II MHC I
DendritischeZelle Ag-Prozessierung
& Präsentation Ag-Prozessierung
& Präsentation
T-Zellrezepror erkennt Fremd (Peptid) und Selbst (MHC)(MHC restiction - Zinkernagel & Doherty )
T-Zellrezepror erkennt Fremd (Peptid) und Selbst (MHC)(MHC restiction - Zinkernagel & Doherty )
Division of Molecular Immunology, Universitätsklinikum Erlangen 21
Merkmale der adaptiven Immunität
Organimsus erinnert sich an Antigen und antwortet mit einer
besseren
spezialisierteren
schnelleren
auf das jeweilge Pathogen zurechtgeschnitte Antikörper-antwort
(über Affinitätserhöhung)
(durch IgH-Klassenwechsel)
(Signalwege ?)
Division of Molecular Immunology, Universitätsklinikum Erlangen 22
Lymphnodes
PeyerPlaques
Spleen
Appendix
Tonsils
Anatomical Location
Secondary lymphatic
organs
Afferent lymphatic
vessel
Paracortex(T cell zone)
B cell zone
medulla
arteria
SecundaryB cell follicle
„Germinal center“
PrimaryB cell
follicule
Efferentlymphaticvessel
venule
From Janeway
Primaryfollicle Secondary
follicle +
Germinal center
CXCR5
IgM
Antigen
+/-TH
ExpansionIgM
Naive B cell B cell
focus
Short-livedPlasma cellShort-livedPlasma cell
Long-lived plasma cellsLong-lived
plasma cells
Memory B cell
Memory B cell
IgGIgAIgE
IgGIgAIgE
Anatomy of B Cell Response
IgD - B cellsPNA - GC B cellsCD3 - T cells
Spleen section - 7 days SRBC
BT
HEV
GC
T C
ell Z
on
eB
Ce
ll Z
on
e
+TH
Division of Molecular Immunology, Universitätsklinikum Erlangen 24
T-Zell-abhängige B-Zellaktivierung
TH
Lymphknoten
DendritischeZelle (DZ)
NaiveCD4-T
NaiveCD8-T
MHC II MHC IDZ
TK
MHC II
T-Zellzone (extrafollikulär)
B
Infektion
TFH
Plasmazelle
Gedächtnis-B
B-Zellzone (follikulär)
Primaryfollicle
IgM
Antigen
+TH
ExpansionIgM
Naive B cell B cell
focus
Short-livedPlasma cellShort-livedPlasma cell
Anatomy of B Cell Response
IgD - B cellsPNA - GC B cellsCD3 - T cells
Spleen section - 7 days SRBC
BT
HEV
GC
T C
ell Z
on
eB
Ce
ll Z
on
e
Division of Molecular Immunology, Universitätsklinikum Erlangen 26
Extrafollikuläre B/T-Zell-Kooperation
CD40LBZR
Immunologische Synapse
CD40
IL2/4/5 ILR
12
MHC II +
PeptidTZRTH
B+
Gekoppelte Erkennung(Linked recognition)
B- und T-Zellepitop müssen auf dem gleichen Molekül liegen
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
B-Zell-Epitop(Peptid oder
Hapten)
TrägerT-Zell-Epitop
(Peptid)NO2
OH
Träger
Primed TH
http://www.studon.uni-erlangen.de/crs816430.html
STUDON
RINGVORLESUNG
Primaryfollicle Secondary
follicle +
Germinal center
CXCR5
IgM
Antigen
+/-TH
ExpansionIgM
Naive B cell B cell
focus
Short-livedPlasma cellShort-livedPlasma cell
Long-lived plasma cellsLong-lived
plasma cells
Memory B cell
Memory B cell
IgGIgAIgE
IgGIgAIgE
Anatomy of B Cell Response
IgD - B cellsPNA - GC B cellsCD3 - T cells
Spleen section - 7 days SRBC
BT
HEV
GC
T C
ell Z
on
eB
Ce
ll Z
on
e
+TH
Division of Molecular Immunology, Universitätsklinikum Erlangen 29
GC
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
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
Germinal Center (dt.: Keimzentrum)
Elise PunkenburgBachelorarbeit, Erlangen 2008
30
Light Light zonezone
Dark Dark zonezone
TFH
Germinal Center
Germinal Center
CSR
SHM
Selection
“Memory”plasma cell(long-lived)
“Memory”plasma cell(long-lived)
Memory B cell
Memory B cell
IgG, IgA, IgE
IgG, IgA, IgE
Expansion
TFHT cell T cell zonezone
B cell B cell zonezone
Germinal Center ReactionModified from McHyzer-Williams2011
FDC
??
CSR?
GC exit
IgM
31GC - Molecular Changes at the Ig locus
1. Somatic hypermutation
2. IgH class switch
IgM IgG, IgA, IgE
VH
CH
VL
CL
Better and more specialized antibodiesBetter and more specialized antibodies
AID
Division of Molecular Immunology, Universitätsklinikum Erlangen 32
Kinoshita & Honjo NRCB(2001)
IgH Class Switch Recombination (CSR)
Division of Molecular Immunology, Universitätsklinikum Erlangen 33
IgM
IgG1
CSR
Cytokine
VH CCC3 C1 C2b C2a CC
SS3 S1 S2b S2a SS S, switch regions
DNA-Looping-out und Deletion
VH C1 C2b C2a CC Switch circle
IgH Class Switch Recombination (CSR)
AID
IL4LPS
Jäck et al.,P.N.A.S. USA 1988von Schwedler et al., Nature 1990
• Synapsis• Incision• Double-strand breaks• End-Joining/Ligation
VH C1 C2b C2a CC
C
C
C3
Division of Molecular Immunology, Universitätsklinikum Erlangen 34
Einfügen von Punktmutationen willkürlich über das gesamte V-Exon des L- und H-
Ketten gens verteilt
V
SHM und Ig-Mutator
Somatic Hypermutation (SHM) of V Regions
AID
Jacob et al., Nature 1991
35Germinal Center Reaction - Selection
Light Light zonezone
Dark Dark ZoneZone
Selection
Follicular dendritc cell
(FDC)FcR
CR
native antigen
C
B cellwith high-affine
Ag receptor
Division of Molecular Immunology, Universitätsklinikum Erlangen 36
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
Affinity Maturation
PROBLEM: Self-reactive B cells could be selected by self-antigen on FDC – ► Requirement for another checkpoint
PROBLEM: Self-reactive B cells could be selected by self-antigen on FDC – ► Requirement for another checkpoint
37
Light Light zonezone
Dark Dark zonezone
TFH
GC reactionGC reaction
CSR
SHM
Selection
Expansion
TFHT cell T cell zonezone
B cell B cell zonezone
Germinal Center ReactionModified from McHyzer-Williams2011
FDC
CSR?
AID
IgM
SelectedB cell
Division of Molecular Immunology, Universitätsklinikum Erlangen 38
B/T-Kooperation im Keimzentrum
Nutt & Tarlinton, Nat Immunol. 2011
39
Light Light zonezone
Dark Dark zonezone
TFH
GC reactionGC reaction
CSR
SHM
Selection
“Memory”plasma cell(long-lived)
“Memory”plasma cell(long-lived)
Memory B cell
Memory B cell
IgG, IgA, IgE
IgG, IgA, IgE
Expansion
TFHT cell T cell zonezone
B cell B cell zonezone
Germinal Center ReactionModified from McHyzer-Williams2011
FDC
??
CSR?
AID
GC exit
IgM
SelectedB cell
40
Staudt/
Calame/
Lassila
Model
Modified from Nutt et al., 2011
Control of PC Differentiation
Pax5
MiTF
Bcl6
Bach2
GC Program
IRF4 ↑AID
Repair
PC Program
Xbp1
Blimp1
IRF4↑↑↑↑
41
Light Light zonezone
Dark Dark zonezone
TFH
GC reactionGC reaction
CSR
SHM
Selection
“Memory”plasma cell(long-lived)
“Memory”plasma cell(long-lived)
Memory B cell
Memory B cell
IgG, IgA, IgE
IgG, IgA, IgE
Expansion
TFHT cell T cell zonezone
B cell B cell zonezone
Germinal Center ReactionModified from McHyzer-Williams2011
FDC
??
CSR?
AID
GC exit
IgM
Division of Molecular Immunology, Universitätsklinikum Erlangen 42
Plasma cell
MemoryB cell
Ag
IgM
IgD
NaiveB cell
IgGIgAIgE
IgA
IgM
IgG, IgE
Effector B Cells
• Igmem → Igsec
• Ig production ↑ (100x)
• Long-lived PC in bone marrow niches
Plasma Cell (PC)
• IgGmem, IgAmem or IgEmem
• Reacts faster to Ag
• Circulates through body
• Long-lived (does not require antigen contact for survival)
• Generation requires T help
Memory B Cell
Division of Molecular Immunology, Universitätsklinikum Erlangen 43
Ag
IgM
IgD
NaiveB cells
Short-livedPlasma cells
IgM
+/-TH
DifferentiationClonal Expansion
+/-TH
MemoryB cell
IgG, IgA, IgE
+TFH
Long-lived plasma cells+TFH
Summary: Effector B Cells
IgM
Germinal centerreaction
• Proliferation
• Somatic hypermutation
• Selection
• class switch
• Effector cells
IgG, IgA, IgE
Better and more
specialized abs
AID
Adaptive Humoral Immunity
AIDActivation-Induced Deaminase
Master regulator of secondary antibody diversification
and ?????
Division of Molecular Immunology, Universitätsklinikum Erlangen 45
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)
→ Activation-Induced Deaminase = AID (Gensymbol AICD)
Notwenig für CSR und SHM
AIDCytosin Uracil
AID - Entdeckung
Deamidase
R C
O
NH2
R C
O
OH
H2O NH3
Säureamid Carbonsäure
Division of Molecular Immunology, Universitätsklinikum Erlangen 46
Kurzer Ausflug in APOBEC-Familie
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: → Editieren von RNA und DNA→ Immunität gegen Viren→ Inaktivierung von Retroelementen
Mensch
Goila-Gaul andStrebel,Retrovirology 5:51, 2008
Division of Molecular Immunology, Universitätsklinikum Erlangen 47
N C
Apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1
AUG CAA UAG APOB mRNA
APOB100
Leber
AUG UAA UAG Mut APOB mRNA
N C APOB48• Navaratnam et al., LBC 1993
• Teng et al., Science, 1993
DuodenumAPOBEC1
(one !!!! mutation in RNA)
APOBEC1 – Prototyp eines RNA-editierenden Enzyms
Division of Molecular Immunology, Universitätsklinikum Erlangen 48
APOBEC3G – Innate Abwehr gegen Viren und Transposon
Nur in Säugern (Lymphozyten)
Mutiert neuen ss-cDNA-Strang
Modelle antiviraler Wirkmechanis-men von APOBEG3:
(1) Reduziert Bildung viraler Transkripte
(2) Inaktivierende Mutationen in Virions
(3) Induziert Abbau mutierter Trankripte
(4) Induziert Abau nicht-mutierter Trans- kripte durch Rekrutierung zellulärer Nukleasen →Editing-unabh.Mechanismus
RNA (viral oder Retrotransposons)
RT APO3
X XX XX
mutiertecDNA
Sheehy et al. Nature 2002
Division of Molecular Immunology, Universitätsklinikum Erlangen 49
Kurzer Ausflug: Retroelements
Endogene Retroelemente (bis zu 45% des menschlichen Genoms) Mutatoren, werden aber auch selber mutiert
Biologische Aktivität/Funktiono Genduplikationen
o Vergrößerung des Genoms
o Insertionsmutagenese
new protein
new RNA new cDNA
insertional mutagenesis
AIM2TLR9APOBEC3Trex1AID
TLR7RIG IMDA5
AdaptiveImmunity
From M. Wabl
Division of Molecular Immunology, Universitätsklinikum Erlangen 50
AID – A Hypermutator
AIDCytosin 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 51
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 52
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
AID: How does it work?
• Constans, A. Class /Switch Wars 2004. The Scientist18(18):28• Honjo et al. 2004. Immunity 20:659-68
Division of Molecular Immunology, Universitätsklinikum Erlangen 53
N C
AUG CAA UAG „Mutator“encodingmRNA
Aktiver „Mutator“(Endonuklease)
AID
AUG UAA UAG
Inaktiver MutatorN C
AID↑ in Keimzentrums-B-Zelle
RNA-Editing-Hypothese
„Mutator“encodingmRNA
Division of Molecular Immunology, Universitätsklinikum Erlangen 54
RNA-Editing-Hypothese
Hinweise AID hat sehr starke Sequenzähnlichkeit mit APOBEC1 (RNA-
Editierung)
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)
55
CV exon C
C C
Base excision and error prone
Base excision and error prone
Base excision and mismatch repair
Base excision and mismatch repair
A G
A G
CC C CC C
SHMSHM CSRCSR
U U UU U UU U
DNA Model
Division of Molecular Immunology, Universitätsklinikum Erlangen 56
Ausflug – DNA-Reparatur
• http://www.web-books.com/MoBio/Free/Ch7G.htm• Peterson and Cote, G&D, 2004
Three major DNA repairing mechanisms: Base excision (BER), nucleotide excision (NER) and mismatch repair (MMR).
Division of Molecular Immunology, Universitätsklinikum Erlangen 57
• 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-N-glycosidase = 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.
DNA-Reparatur – Base excison Repair (BER)
Division of Molecular Immunology, Universitätsklinikum Erlangen 58
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 59
DNA-Reparatur – Mismatch Repair (MMR)
• Important for mutations unable to be repaired by BER or NER
• 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.
E.coli
Dam methylates template at GATC
GATC
Division of Molecular Immunology, Universitätsklinikum Erlangen 60
BER
• AID deaminates C to U at accessible sites in S regions
• Multiple sites of deamination and UNG/APE-induced nicks
• Break processing (Mismatch repair = MMR)
• Gaps and DSBs produced
• DNA synthesis up to nick• Blunt ends at DSBs• S region ligation
From: Stavnezer et al., Annu. Rev. Immunol. 2008
MMR
nicks
nicks
DSB
Blunt DSB
DNA-Mutations-Modell (CSR)
CSR reduced by 95 % if BER is deficient!
Division of Molecular Immunology, Universitätsklinikum Erlangen 61
In vitro, AID mutates ssDNA but not RNA
Class switch recombination is inhibited (95%) and somatic hypermutation is perturbed in UNG-(BER) deficient mice
DNA-Mutations-Modell - Hinweise
Division of Molecular Immunology, Universitätsklinikum Erlangen 62
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 HEK-Zellen (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 63
ADAPTIVE IMMUNITY
Formation of 2 antibody repertoire
AID targets Ig locusCytosine Uracil
Protection from
Autoimmune disease
Proliferative disease
nucleus
INNATE IMMUNITY ?
cytoplasm
Activated B cell
AID – Zusammenfassung
CSR SHM
Host response to
viral infection
Inhibition of retroelement
activity
M. MetzerPromotion 2010
Division of Molecular Immunology, Universitätsklinikum Erlangen 64
IgG
IgM
NaiveB-Zelle
LanglebigePlasmazelle
(IgG, IgA, IgE)
LanglebigePlasmazelle
(IgG, IgA, IgE)
Gedächtnis-B-Zelle
(IgG, IgA, IgE)Keim-
zentrums-reaktion
IgGt (Tage)
Log
CA
K im
Ser
um
Primärantwort Sekundärantwort
3
2
1
0 4 8
IgM
10Ag 20Ag
Schneller und mehr !!!!
IgM
0 4 8
LanglebigePlasmazelle
(IgG, IgA, IgE)
Gedächtnis-B-Zelle
(IgG, IgA, IgE)Keim-zentrums-reaktion
KurzlebigePlasmazelle
(IgM)Ag+TH
IgMKurzlebigePlasmazelle
(IgM)
NaiveB-Zelle
Ag+TH
Übersicht: Adaptive humorale Immunität