Transcript Kein Folientitel

PhD Education Program FB14 and FIRST
Selected Topics in Molecular Medicine
and Basic Research
Frankfurt, November 26, 2007
Humoral Immunity
Hans-Martin Jäck
Division of Molecular Immunology
Department of Internal Medicine III
Nikolaus-Fiebiger-Zentrum
University of Erlangen-Nürnberg
Universitätsklinikum
Erlangen
Topics
 Overview: Immunity
 Discovery of humoral immunity
 B cell maturation and antibody repertoire
 Generation of protective antibodies
http://www.molim.uni-erlangen.de/downloads/
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Overview - Immunity
• Immune system
The immune system is a defense machinery of living organims against pathogens and foreign
substances, also known as antigens
• Immunity
The components of the immune system create barriers, thereby protecting any individual
against pathogens (Immunitas, lat.: ‚BEFREIUNG VON LASTEN‘)
Barriers againts Pathogens
•
Mechanical barriers
(skin, hair)
•
Chemical barriers
(acidic pH in stomach)
•
Immunological barriers
(cells, antribodies and
mediators)
Division of Molecular Immunology, Universitätsklinikum Erlangen
Inherited
Immunity
Aquired
Immunity
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The Two Branches of the Immune System
Inherited Immunity
Aquired Immunity
(Adaptive Immunity)
(Innate Immunity)


Very old (plants, animals)
Already present


→ fast response (hrs)




Recognition of chemical
patterns
Few pattern recognition
receptors (TLR)
No memory
Complement, Macropahes
Only in vertebrates
Inducible
→ slow response (days)




Specific recognition of
defined structures
Millions of specific
antigen receptors
Memory
B- and T cells
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Adaptive Immunity – Organs
Tonsilen
Lymphknoten
Thymus
Primäre lymphatische Organe
 Reifung und Selektion funktioneller
Lymphozyten
Milz
Appendix
Peyer'sche
Plaques
Knochenmark
Sekundäre lymphatische Organe

LymphKnoten
Induktion der spezifischen, adaptiven
Immunantwort (Generierung von
Effektorzellen)
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Adaptive Immunity – Cell Development (primary lymphatic organs)
Bone marrow, fetal liver
Hematopoietic
stem cell
Thymus
B cells
CD4+
T cell
CD8+
T cell
Monocyte
Erythocyte
Blood, spleen, lymph node, gut e.t.c
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Adaptive Immunity – Induction (in peripheral lymphatic organs)
Antigen Receptors
Specific components
From place of infection through
lymph or blood vessels to
lymphatic organ
Naive
Cells
B
T (CD4)
Dendritic
cell
T (CD8)
BCR
Effector
cells
Plasma
Effector
Mediators
THelper
Ag
TKiller
Lymphokines
TCR
Humoral
Immunity
Cellular
Immunity
MHC II
Division of Molecular Immunology, Universitätsklinikum Erlangen
MHC I
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T-B-Cooperation (linked recognition)
IL2/4/5
Antigen
with
epitopes
ILR
1
CD40
2
CD40L
B
TH
TCR
BCR
 Proliferation
 Differenzierung
MHC II
+
Peptid
CD40/CD40L deficiency
→ Hyper-IgM Syndrome I
Immunologic
synapse
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Discovery of Humoral Immunity
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Discovery of Humoral Immunity (Emil von Behring 1890)
Inactivierted
Tetanus
Serum
10

Pathogenic
Tetanus
20
Inactivierted
Diphteria
Serum

10
Emil von Behring (1890)
• Soluble, onducibel and specific immunity trough immunisation with pathogens
• Individual can be protected by transferring serum from immunized animal
→ 1st Nobel Price 1901 for Serum Therapie
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Models und Chemotherapy (Paul Ehrlich 1900)
• Ehrlich‘s Sidechain Theory (1900)
(Explains inducibilIty and specificity)
• Nobel price 1906 for chemotherapy
(Salvarsan against Syphillis)
Seitenkette
Toxin
Lösliche Seitenketten
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Modelle und Chemotherapie (Paul Ehrlich 1905)
• Seitenketten-Theorie (1900)
(Explains inducibilIty and specificity)
• Nobelpreis 1906 for Chemotherapie
(Salvarsan against Syphillis)
Seitenkette
Toxin
Lösliche Seitenketten
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From the Seitenketten Model to Clonal Selection
From Ehrlich‘s Seitenketten-Theorie (1900) to Burnet‘s, Jerne‘s & Talmage‘s
clonal selection (1956-58)
Antigen (Ag)
(Antikörper
generierend)
B cell
receptor
(BCR)
+TH
Memory
B cell
B cells
B cell
clon
+/- TH
IgG
IgA
IgE
Antibodies
Plasma
cell
(monospecific)
clonal expansion
differentiation
(Multiplication)
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Antibody structure (Edelman, Porter, Hilschmann & Craig 1962-69)
V
G. Edelman
L-Kette
C
L
N. Hilschmann
L
H chain
R. Porter
1962
1972
L. Craig
Quaternary Structure (H2L2)
Nobel prize in Medicine
1965
Discovery of V regions
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Duale Funktion der Antikörper – Magische Kugeln
Spezifität
VH
CH1
VL
CL
L-Kette
CH2
CH3
H-Kette
• Biochemische
Eigenschaften
 Gewebeverteilung
 Halbwertszeit
• Biologische
Effektorfunktion
 Komplement
 Opsonisierung
 Rekrutierung
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Ig Classes establish antibody effector repertoire
Human and mouse have five Ig classes
IgM
Antigen receptor on naive B cells
IgG
Major antibody in blood, and thus of ‚internal‘ defense
IgA
Major antibody on mucosal surfaces, and thus of external defense
IgE
Mediates activation of effector cells during worm infection, but
also responsible for allergic reactions (dust mites, asthma, hey
fever)
IgD
Circulating antibodies with no function
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B-Zellreifung und AK-Repertoire
Knochenmark
z.B. Milz
Plasmazelle
Ag
Stammzelle
Pro-BZelle
VH →D→JH
Frühe
Prä-B
Späte
Prä-B
VL → JL
Unreife
B-Zelle
Reife
B-Zelle
GedächtnisB-Zelle
Primäres B-ZellRepertoire
~109-1012 Antikörper
Division of Molecular Immunology, Universitätsklinikum Erlangen
Vettermann et al.
Sem. Immunol, 2006
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Somatic Ig Gene Rearrangements
VH
CH
Nobel Price
1987
Membane IgH
CH-Exons
VH-Exon
DNA in B cell
DNA in stem cell
VH
D
JH
Functional immunglobulin genes are assembled from gene segments by DNA recombination
during B cell maturation from hematopoietic stem cells (Susumu Tonegawa, 1976)
V(D)J recombination generates Ab Diversity
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Antibody REPERTOIRE
13 DH

4 JH

5 CH

~ 85 Vκ

4 Jκ

1 Cκ
VH Regions
(ca. 6760)
Recombinatorial
Vκ Regions
(340)
Recombinatorial
CH2

Antigen binding site
= Paratop
“Magic Part”
CH3
 ~ 134 VH
Combinatorial diversity
(reperire, lat.
wiederfinden)
~ 2.3 x 106 Ab
Junctional
diversity
109 - 1012 Ab
Division of Molecular Immunology, Universitätsklinikum Erlangen
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V(D)J Recombination – Genetic mechanism
Vκ1
Jκ
Vκ n
Looping-out (RAG1/2)
Cκ
DNA in
stem cell
Recombination signal sequence
(hepatmer-spacer-nonamer)
(Signal Joint)
Deletion
(RAG1/2)
Ligation
(NHEJ)
(Coding Joint)
Division of Molecular Immunology, Universitätsklinikum Erlangen
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V(D)J Recombination – Biochemistry
Schritt 1: Erkennung und Paarung von RSS
(DNA-bindende Proteine)
Schritt 2: Schneiden → Doppelstrangbrüche
(Endonukleasen)
Schritt 3: Prozessieren der Enden
(Polymerasen, Endo- und Exonukleasen)
Schritt 4: Ligieren der Enden
(Ligasen)
Nur in
Lymphozyten
(RAG1/2-Proteine)
Alle Zellen
„Doppelstrangbruchreparaturproteine“
(NHEJ = Non-homologues
end joining)
Division of Molecular Immunology, Universitätsklinikum Erlangen
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V(D)J Recombination – Defects
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Quality Control of B Cell Maturation
Kontrollpunkte
Paarung von
Ig-Ketten?
Autoreaktivität?
Plasmazelle
Ag
Stammzelle
Pro-BZelle
Frühe
Prä-B
Späte
Prä-B
Unreife
B-Zelle
Reife
B-Zelle
GedächtnisB-Zelle
VL → JL
VH →D→JH
Pre-BCR
BCR
Division of Molecular Immunology, Universitätsklinikum Erlangen
Vettermann et al.
Sem. Immunol, 2006
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Immature and Mature Ig Receptors
unique
tails
VpreB
LC
l5
β8
VH
VH
Cµ1
Cµ1
Cµ2
µHC
Cµ2
µHC
Cµ3
Igα/Igβ
Cµ3
Igα/Igβ
Cµ4
Cµ4
Pre-B Cell Receptor
B Cell Receptor
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Function of the Pre-BCR
 Ankurbeln des Zellzyklus
 Vervielfältigung von Prä-B-Zellen mit
paarungsfähigen µH-Ketten durch
selektive klonale Expansion
µH
Igαβ
 Überlebenssignale
 Umleitung der V(D)J-Rekombinase
vom IgH- zum IgL-Lokus ???
 Allelausschluss am IgH-Lokus
Pre-BCR
 Öffnen des IgL-Lokus
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Pre-B cell expansion increase combinatorial repertoire
BCR
VH1
VH1VL1
VH1
VH1VL2
VH1
VH1VL3
VH1
VH1VL4
Pre-BCR
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Pre-BCR Signal Initiation
Aus Vettermann et al, Sem Immunol, 2006
Pre-B cell autonomous
Stroma cell-dependent
Ligand-independent
Ligand-dependent
BM stroma cell
pre-B cell
pre-B cell
pre-B cell
pre-B cell
B
C
D
E
Signals induced
by self-ligand
Signals induced
by external ligand
Signals induced by
receptor assembly
Signals induced by
receptor self-ligation
The l5 Unique Region distinguisges a pre-BCR from a BCR, and thus
allows pre-BCR-specific signal enhancement
Division of Molecular Immunology, Universitätsklinikum Erlangen
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„Eliminierung“ autoreaktiver B-Zellen
Zentrale Mechanismen
• Deletion
• Anergie
• Editieren des Rezeptors
Editieren des Rezeptors
VL
VJ
JL
CL
Periphäre Mechanismen
• Ignoranz
• Anergie
• Kompetition
Autoreaktiver
Rezeptor
Division of Molecular Immunology, Universitätsklinikum Erlangen
Editierter
Rezeptor
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B-Zellreifung und AK-Repertoire
Knochenmark
z.B. Milz
Plasmazelle
Ag
Stammzelle
Pro-BZelle
VH →D→JH
Frühe
Prä-B
Späte
Prä-B
VL → JL
Unreife
B-Zelle
Reife
B-Zelle
GedächtnisB-Zelle
Primäres B-ZellRepertoire
~109-1012 Antikörper
Division of Molecular Immunology, Universitätsklinikum Erlangen
Vettermann et al.
Sem. Immunol, 2006
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Reife B-Zellpopulationen
• B1-Zellen
• B2-Zellen
– Follikuläre B-Zellen
– Marginal-Zonen B-Zellen
Division of Molecular Immunology, Universitätsklinikum Erlangen
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B1 und Follikuläre B2-Zellen
• B1-Zellen
•
•
•
•
•
•
In Peritoneal- und Pleuralhöhle
Entstehen schon während der Embryonalentwicklung
Selbsterneuernd
T-Zell-unabhängige schnelle Antikörperproduktion
Dienen der Abwehr auf mukösen Oberflächen
Markers: CD5+ und IgM↑
• Follikuläre B2-Zellen
• In allen lymphatischen Organen
• „Normale“ rezirkulierende B-Zellen
• Markers: IgM↓, IgD↑, CD21/35↑, CD23↑
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Marginalzonen(MZ)-B2-Zellen
• Residente, langlebige Zellen in
der Marginal-Zone der Milz
• Sehr gute Immunantwort gegen
Polysaccharide (Kapselbakterien)
• Vermitteln „frühe“ Immunabwehr
gegen Erregern, die aus dem
Blut in die Milz gelangen
• Nach Kontakt mit Antigen innerhalb von 4 Stunden Differenzierung in Plasmazellen
• Markers: IgM↑, IgD↓, CR1/2↑,
CD23↓, CD1d↑ (non-klassisches
MHC)
Milz mit Ausschnit zeigen
Cyster, Nat. Immunol. 2000
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Establishment of Humoral Immunity
T-Zell-Zone
Extrafollikuläre
AntigenReaktion
(Antikörper
generierend)
Reife
B-Zelle
IgM
+/-TH
+/- TH
+TH
B-Zell-Zone
Expansion Differenzierung
Kurzlebige
Plasmazelle
(IgM)
B-ZellKlon
Keimzentrumsreaktionen
• Gedächtnis
• IgH-Klassenwechsel
GedächtnisB-Zelle
(IgG, IgA, IgE)
• Selektion
Primärfollikel
Lymphknoten
SekundärFollikel mit
Keimzentrum
• Somatische
Hypermutation
• Proliferation
Division of Molecular Immunology, Universitätsklinikum Erlangen
Langlebige
Plasmazelle
(IgG, IgA, IgE)
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The World of Antigens (Antikörper generierend)
B cells recognize everything
•
•
•
•
•
Proteins
Lipids
Nucleic acids
Carbohydrates
Chemical molecules
or haptens (half antigens)
• Even plastic
But only proteins are good thymusdependent antigens
Division of Molecular Immunology, Universitätsklinikum Erlangen
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T-B-Cooperation (linked recognition)
IL2/4/5
ILR
1
CD40
2
CD40L
B
TH
TZR
BZR
 Proliferation
 Differenzierung
MHC II
+
Peptid
Immunologische
Synapse
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Follicular reaction (in germinal center)
Apical light zone
• IgH class switch
• Memory B
• Plasma blasts
Memory B
Plasmablast
Long-lived
plasma cells
in bone marrow
Selected centrocyte
Basal light zone
B
T
Janeway
Follicular dendritic cell (FDC)
Ag-Ab complexes
Centrocyte
Selection of highaffine centrocytes
by binding to AgAb complexes on
FDC
Tingible-body
macrophage
Dark zone
Somatic mutation
of VH / VL regions
Kuby
Centroblast
(activate B cell)
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Somatic Hypermutation (SHM)
Distribution of mutations before and after selection by Ag-Ab complexes on FDC
Mutations cluster in paratop (Ag binding sites)
AID
VH
residue
Selection
Mutated GC centrocystes before selection
o bind immunogen
 not at all
 with lower affinity
 with the same affinity
 with the same affinityt
 with higher affinity
o bind different antigen
o react with self-antigen
o produce no BCR (nonsense Mutation)
Mutationsrate von IgV
= 10-5 bp/Generationszeit
Natürliche Mutationsrate = 10-12 bp/Generationszeit
Somatic hypermutation
Division of Molecular Immunology, Universitätsklinikum Erlangen
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AID, a critical component of the Ig hypermutator
 AID = Activation-Induced Deamidase
• Only expressed in activated
germinal center (GC) B cells
AGCT
TCGA
(cDNA was cloned by a substractive cDNA approach
from IL-activated B cell line)
AID
• Converts C to U (oxydative deamination)
Cytosin
AID
AGCT
TUGA
Uracil-Nglycosidase
(UNG)
Uracil
 Homology to RNA editing enzyme APOBEC
AGCT
T GA
Modifier (??)
fills gap with
any base
 AID knock-out mice and AID-def. patients
• no somatic hypermutation (SHM)
• no IgH class switch recombination (CSR)
→ Hyper-IgM-Syndrome Type II
SHM
Division of Molecular Immunology, Universitätsklinikum Erlangen
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IgH Class Switch Recombination (CSR)
VH
Cm
Cd
Cg3
Sm
IgM
Sg3
Cg1
Sg1
Cg2b Cg2a
Sg2b
Sg2a
Ce
Se
Ca
Sa
Cd
Cm
CSR
VH
Cg3
Cg1
Cg2b Cg2a
Ce
Ca
Cg2b Cg2a
Ce
Ca
AID
VH
Cg1
IgG1
Molekulare Grundlage für die Erzeugung der Repertoires der biologischen Effektorfunktionen durch Austausch der
konstanten Regionen der H-Kette unter Beibehaltung der Antigenbindungsstelle (nur bei Ig aber nicht bei TZR)
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Terminal plasma differentiation
Requirement for BLIMP-1 (B lymphocyte induced maturation protein 1) and XBP-1 (X-box
binding protein), two transcriptional suppressors/repressors
Transfection of Blimp1 gene
into lines and primary B cells
Blimp1 +
• Calame and coworkes
• Davis and coworkers
B cell
Knock-out mouse
(RAG complementation
Approach)
XBP-1 -/-
Glimcher and coworkes
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Antikörper-Effektorreaktionen
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Summary: The Antibody Repertoire
Antigen-induced B Cell activation (Periphery)
B Cell Maturation (BM)
Ag
+TH
Germinal
Center
Plasma cell
• SHM
IgG
IgA
IgE
• CSR
• Memory B
• Plasma
Memory B cell
Primary repertoire
V repertoire - specificity
Modified secondary repertoire
V repertoire - increased affinity
CH repertoire - effector functions
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Summary: Primary and Seconday Ab Response
Primary Response
• Naive B
• Predominantly IgM but
also other classes
• Lag 7-10d
• Peak 4-7d
• Thmyus-dependent &
-independent Ag
• Low affinity (~ 10-7 1/M)
Secondary Response
• Memory B
• IgG and others
• Lag 1-3d
• Peak 3-5d
• Thymus-dependent Ag
• Higher affinity (10-10 1/M)
• Higher titers
(100 – 1000x)
Division of Molecular Immunology, Universitätsklinikum Erlangen
Aus Kuby
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