Kein Folientitel

1
Vertiefungsmodul Immunbiologie Ringvorlesung Erlan
gen ? 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
2
Plasma Cell Differentiation
Memory B cell
Naive B cells
Ag TH
More affine specialized antibodies
Germinal Center Reaction AID
Memory Plasma cell
3
THEMEN

• Überblick Adaptive humorale Immunität
• Keimzentrumsreaktion, Affinitätsreifung und
  IgH-Klassenwechsel
• AID und APOPECs
• Funktion und Wirkmechanismus von AID
• AID und angeborene Immunität

4
Immunsystem bildet Barrieren
Angeboren
Keime fremde Substanzen
Erworben

• Physikalisch
• Haut
• Schleim
• Darmflora
• Flimmerhaare
• Physiologisch
• pH
• Temperatur
• Zellulär (Leukozyten)
• Makrophagen
• Granulozyten
• Lymphoyzen
• Entzündung
• Die vier ors

• B- und T-Zellen
• Antikörper
• T-Zell-Botenstoffe
• Interleukine
• Zytokine
• Lymphotoxine
• Chemokine
• Gedächtnis !!!!
• Adaptive Immunität

• Bakterien
• Pilze
• Würmer
• Viren
• Fremde Eiweiße

BARRIEREN
Abwehrsystem IMMUNSYSTEN (Immunitas, lat.
Freisein von Leistungen/Lasten)
5
Adaptive humorale Immunität
6
Anatomie der Adaptiven Immunität
TFH
Infektion
7
Antibodies Structure and Function

• 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 k und l 5 CH m, d, g, a und e (Isotyp)

Antigenbindung
Effektor- funktion
IgM
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Antibodies Structure and Function
Antigen binding sites Paratop Magic Part
VH
VL
CH
CL
CH
CH
CH
Antibodies are bifunctional (Paul Ehrlichs Magic
Bullets)
9
Antibodies Effector Functions

• Neutralisation
• Agglutination
• Activation of Complement
• Enhancement of Phagocytosis (Opsoniation)
• Recrutement of effector cells (Neutrophils,
  natural killer cells)

10
Adaptive Humoral Immunity Generation of the
Primary B Cell Repertoire
B cell receptor 1
Antigen 1
One B cell - One Receptor
11
Maturation of B Cells
Central Maturation(Bone marrow)
Pre-BCR
BCR
Late Pro-B
Late Pre-B
Immature B cell
Early Pre-B
Stem cell
VH?D ? JH
VL ? JL
L
H
12
Generation of Antibody Diversity
S. Tonegawa Nobel Price 1987 Basel Institute of
Immunology
Recombination
B cell
VH Exon
Transcription Translation
VH C?
B cell
N C
µH chain
V(D)J recombination generates antibody diversity
13
Summary Preimmune Repertoire
4JH
C?
134 VH
13 D

• Recombinatorial diversity
• Random assembly from V, D J

• Combinatorial diversity
• 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 Mensch Maus Anzahl 1012 109 Neu/Tag
109 106
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HUMORAL IMMUNE RESPONSE
Central Maturation(Bone marrow)
Peripheral Maturation (Spleen)
Pre-BCR
BCR
Late Pro-B
Late Pre-B
Immature B cell
Early Pre-B
Stem cell
Transitional B cells
Mature B cell
VH?D? JH
VL ? JL
L
H
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HUMORAL IMMUNE RESPONSE
Central Maturation(Bone marrow)
Peripheral Maturation (Spleen)
Effector Phase (lymph node, spleen, etc.)
Plasma cell
Pre-BCR
BCR
Ag TH
GC
Late Pro-B
Late Pre-B
Immature B cell
Early Pre-B
Stem cell
Transitional B cells
Mature B cell
Memory B cell
VH?D? JH
VL ? JL
L
H
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Adaptive Humoral Immunity Generation of
Effector B Cells
IgG
Plasma cell
IgM
Ag TH
Ag TH
IgD
Memory B
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Anatomie der Adaptiven Immunität
TFH
Infektion
18
Activation of Naive CD4 T Cells
King et al., Annu. Rev. Immunol. 2008
Activation of naive CD4 T cells in T cell zone
B cell help in follicule
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B Cell Antigens
The World of Antigens (Antibody generating)

• 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

Ag
IgM
IgD
Naive B cells
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T Cell Antigens
T-Zellrezepror erkennt Fremd (Peptid) und Selbst
(MHC) (MHC restiction - Zinkernagel Doherty )
Dendritische Zelle
Ag-Prozessierung Präsentation
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 ?)

22
Anatomical Location
Secondary lymphatic organs
Lymphnodes
Appendix
Spleen
Tonsils
PeyerPlaques
From Janeway
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Anatomy of B Cell Response
Spleen section - 7 days SRBC
Antigen
Expansion
IgM
HEV
IgM
T
/-TH
T Cell Zone
B
Naive B cell
Short-lived Plasma cell
B cell focus
TH
GC
IgG IgA IgE
CXCR5
Memory B cell
B Cell Zone
IgG IgA IgE
IgD - B cells PNA - GC B cells CD3 - T cells
Long-lived plasma cells
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T-Zell-abhängige B-Zellaktivierung
TFH
Infektion
25
Anatomy of B Cell Response
Spleen section - 7 days SRBC
Antigen
Expansion
IgM
HEV
IgM
T
TH
T Cell Zone
B
Naive B cell
Short-lived Plasma cell
B cell focus
GC
B Cell Zone
IgD - B cells PNA - GC B cells CD3 - T cells
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Extrafollikuläre B/T-Zell-Kooperation
ILR

• 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

CD40
1
BZR
2

B
MHC II
Primed TH
Peptid
TH
TZR
27
STUDON RINGVORLESUNG
http//www.studon.uni-erlangen.de/crs816430.html
28
Anatomy of B Cell Response
Spleen section - 7 days SRBC
Antigen
Expansion
IgM
HEV
IgM
T
/-TH
T Cell Zone
B
Naive B cell
Short-lived Plasma cell
B cell focus
TH
GC
IgG IgA IgE
CXCR5
Memory B cell
B Cell Zone
IgG IgA IgE
IgD - B cells PNA - GC B cells CD3 - T cells
Long-lived plasma cells
29
Germinal Center (dt. Keimzentrum)
1884 Fleming discovers germinal centers. The
name GC is based on Flemings 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 PunkenburgBachelorarbeit, 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
30
Germinal Center Reaction
Modified from McHyzer-Williams 2011
Germinal Center
B cell zone
Light zone
TFH
FDC
IgG, IgA, IgE
Memory B cell
Selection
?
GC exit
IgG, IgA, IgE
Dark zone
Memory plasma cell (long-lived)
Expansion
SHM
CSR?
CSR
TFH
IgM
T cell zone
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GC - Molecular Changes at the Ig locus
IgM
IgG, IgA, IgE
VH
AID
VL
CL
CH

1. Somatic hypermutation
2. IgH class switch

Better and more specialized antibodies
32
IgH Class Switch Recombination (CSR)
Kinoshita Honjo NRCB(2001)
33
IgH Class Switch Recombination (CSR)
DNA-Looping-out und Deletion
IL4LPS
VH Cm Cd Cg3
Cg1 Cg2b Cg2a Ce Ca
Sm Sg3
Sg1 Sg2b Sg2a Se Sa
IgM
S, switch regions
Cytokine
AID

• Synapsis
• Incision
• Double-strand breaks
• End-Joining/Ligation

Jäck et al.,P.N.A.S. USA 1988 von Schwedler et
al., Nature 1990
34
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 H-Ketten gens verteilt
35
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
36
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
37
Germinal Center Reaction
Modified from McHyzer-Williams 2011
B cell zone
GC reaction
Light zone
TFH
FDC
Selection
Selected B cell
Dark zone
Expansion
SHM
CSR?
AID
CSR
TFH
IgM
T cell zone
38
B/T-Kooperation im Keimzentrum
Nutt Tarlinton, Nat Immunol. 2011
39
Germinal Center Reaction
Modified from McHyzer-Williams 2011
B cell zone
GC reaction
Light zone
TFH
FDC
IgG, IgA, IgE
Memory B cell
Selection
Selected B cell
?
GC exit
IgG, IgA, IgE
Dark zone
Memory plasma cell (long-lived)
Expansion
SHM
CSR?
AID
CSR
TFH
IgM
T cell zone
40
Control of PC Differentiation
Staudt/ Calame/ Lassila Model
GC Program
Pax5
AID
IRF4 ?
Bcl6
Repair
Bach2
MiTF
Modified from Nutt et al., 2011
41
Germinal Center Reaction
Modified from McHyzer-Williams 2011
B cell zone
GC reaction
Light zone
TFH
FDC
IgG, IgA, IgE
Memory B cell
Selection
?
GC exit
IgG, IgA, IgE
Dark zone
Memory plasma cell (long-lived)
Expansion
SHM
CSR?
AID
CSR
TFH
IgM
T cell zone
42
Effector B Cells
IgG, IgE
IgA
IgM
IgM
Plasma cell
Ag
IgD
IgG IgAIgE
Naive B cell
Memory B cell
43
Summary Effector B Cells
Better and more specialized abs
Ag
IgM
IgG, IgA, IgE
Germinal center reaction
Long-lived plasma cells
IgD
TFH

• Proliferation
• Somatic hypermutation
• Selection
• class switch
• Effector cells

Naive B cells
TFH
Memory B cell
AID
44
Adaptive Humoral Immunity AID Activation-Induced
Deaminase Master regulator of secondary
antibody diversification and ?????
45
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)
• ? Activation-Induced Deaminase AID (Gensymbol
  AICD)
• Notwenig für CSR und SHM

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 and Strebel, Retrovirology 551, 2008
47
APOBEC1 Prototyp eines RNA-editierenden Enzyms
Apolipoprotein B mRNA-editing enzyme catalytic
polypeptide 1
APOB100
N
C
Leber
APOB mRNA
AUG CAA
UAG
Mut APOB mRNA
AUG UAA
UAG

• Navaratnam et al., LBC 1993
• Teng et al., Science, 1993

N C
APOB48
48
APOBEC3G Innate Abwehr gegen Viren und
Transposon

• Nur in Säugern (Lymphozyten)
• Mutiert neuen ss-cDNA-Strang

APO3
RT
X XX XX
RNA (viral oder Retrotransposons)
mutierte cDNA

• 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

Sheehy et al. Nature 2002
49
Kurzer Ausflug Retroelements

• Endogene Retroelemente (bis zu 45 des
  menschlichen Genoms)
• Mutatoren, werden aber auch selber mutiert
• Biologische Aktivität/Funktion
• Genduplikationen
• Vergrößerung des Genoms
• Insertionsmutagenese

From M. Wabl
insertional mutagenesis
TLR7 RIG I MDA5

new RNA
new cDNA
AIM2 TLR9 APOBEC3 Trex1 AID
Adaptive Immunity
new protein
50
AID A Hypermutator

• ? 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

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)

52
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 20659-68

53
RNA-Editing-Hypothese
Aktiver Mutator (Endonuklease)
N
C
AID
Mutatorencoding mRNA
AUG CAA
UAG
AID? in Keimzentrums-B-Zelle
Mutatorencoding mRNA
AUG UAA
UAG
Inaktiver Mutator
N C
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-Aktiv
  ität, 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
DNA Model
SHM
CSR
C?
V exon
Cg
C C
CC
C
CC
C
Base excision and error prone
Base excision and mismatch repair
56
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, GD, 2004

57
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-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. 

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.

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
60
DNA-Mutations-Modell (CSR)

• AID deaminates C to U at accessible sites in S
  regions

CSR reduced by 95 if BER is deficient!
BER
nicks

• Multiple sites of deamination and UNG/APE-induced
  nicks

nicks

• Break processing (Mismatch repair MMR)

MMR
DSB

• Gaps and DSBs produced

From Stavnezer et al., Annu. Rev. Immunol. 2008

• DNA synthesis up to nick
• Blunt ends at DSBs
• S region ligation

Blunt DSB
61
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

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
  3768541867)
• AID-defiziente Patienten haben eine höhere
  Anfälligkeit für Autoimmunsymptome und Leukämien
• ? Wie kann man das erklären?

63
AID Zusammenfassung
Activated B cell
M. Metzer Promotion 2010
nucleus
cytoplasm
ADAPTIVE IMMUNITY
INNATE IMMUNITY ?
AID targets Ig locus Cytosine ? Uracil
Host response to viral infection
Inhibition of retroelement activity
CSR
SHM
? Protection from Autoimmune disease
Proliferative disease
? Formation of 2? antibody repertoire
64
Übersicht Adaptive humorale Immunität
Primärantwort
Sekundärantwort
IgM
Naive B-Zelle
3 2 1
Schneller und mehr !!!!
Log CAK im Serum
20Ag
t (Tage)
0 4 8
0 4 8