Adaptive Humorale Immunität 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 Memory B cell Naive B cells Ag + TH More affine & specialized antibodies Germinal Center Reaction & AID „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

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‘ Bakterien Pilze Würmer Viren Fremde Eiweiße B- und T-Zellen Antikörper T-Zell-Botenstoffe Interleukine Zytokine Lymphotoxine Chemokine Gedächtnis !!!! Adaptive Immunität BARRIEREN Abwehrsystem = IMMUNSYSTEN (Immunitas, lat.: ‚Freisein von Leistungen/Lasten‘)

Adaptive humorale Immunität

Anatomie der Adaptiven Immunität Lymphknoten Naive CD4-T CD8-T MHC II MHC I DZ TK T-Zellzone (extrafollikulär) Dendritische Zelle (DZ) TH Plasmazelle Gedächtnis-B B-Zellzone (follikulär) B TFH Infektion

Antibodies – Structure and Function Antigenbindung 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) VL CL VH CH1 CH2 CH3 L H CHO Effektor- funktion IgM

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 1 2 3 Janeway VL VH VH VL CH CL L H CH Effector sites “Bullet Part” Tissue distribution Serum halflife Complement Phagocytosis Recruting of cells CH CH Antibodies are bifunctional (Paul Ehrlich‘s Magic Bullets)

Antibodies – Effector Functions Neutralisation Agglutination Activation of Complement Enhancement of Phagocytosis (Opsoniation) Recrutement of effector cells (Neutrophils, natural killer cells)

Generation of the Primary Adaptive Humoral Immunity Generation of the Primary B Cell Repertoire B cell receptor 1 Antigen 1 One B cell - One Receptor

Central Maturation (Bone marrow) Maturation of B Cells Central Maturation (Bone marrow) Pre-BCR BCR Stem cell Late Pro-B Early Pre-B Late Pre-B Immature B cell VH→D → JH VL → JL L H

V(D)J recombination generates antibody diversity Generation of Antibody Diversity S. Tonegawa Nobel Price 1987 Basel Institute of Immunology stem cell J segments C V segments D segments ca. 2.5 Mb (mouse) HC locus Recombination kL chain N C VL Exon J V Ck LC locus B cell VH Exon Transcription Translation VH C B cell N C µH chain V(D)J recombination generates antibody diversity

Summary: Preimmune Repertoire 134 VH 13 D 4JH C Recombinatorial diversity Random assembly from V, D & J ca. 107 anti- bodies 109-1012 anti- bodies 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

Peripheral Maturation HUMORAL IMMUNE RESPONSE Central Maturation (Bone marrow) Peripheral Maturation (Spleen) Pre-BCR BCR Stem cell Late Pro-B Early Pre-B Late Pre-B Immature B cell Transitional B cells Mature B cell VH→D→ JH VL → JL Primary repertoire ~109-1012 specificies L H

Peripheral Maturation HUMORAL IMMUNE RESPONSE Central Maturation (Bone marrow) Peripheral Maturation (Spleen) Effector Phase (lymph node, spleen, etc.) Plasma cell Pre-BCR BCR Ag + TH GC Stem cell Late Pro-B Early Pre-B Late Pre-B Immature B cell Transitional B cells Mature B cell Memory B cell Secondary repertoire → Affnity maturation → Effector functions VH→D→ JH VL → JL Primary repertoire ~109-1012 specificies L H

Generation of Effector B Cells Adaptive Humoral Immunity Generation of Effector B Cells Plasma cell IgG IgM Ag + TH Ag + TH IgD Memory B

Anatomie der Adaptiven Immunität Lymphknoten Naive CD4-T CD8-T MHC II MHC I DZ TK T-Zellzone (extrafollikulär) Dendritische Zelle (DZ) TH Plasmazelle Gedächtnis-B B-Zellzone (follikulär) B TFH Infektion

Activation of Naive CD4+ T Cells cytokines Dendritic cell King et al., Annu. Rev. Immunol. 2008 Activation of naive CD4+ T cells in T cell zone B cell help in follicule

B Cell Antigens The World of Antigens (Antibody generating) Clonal Expansion +/-TH Short-lived Plasma cells IgM +/-TH Differentiation 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 Memory B cell IgG, IgA, IgE +TH Long-lived plasma cells IgD Naive B cells

T Cell Antigens T-Zellrezepror erkennt Fremd (Peptid) und Selbst (MHC) (MHC restiction - Zinkernagel & Doherty ) B-Zell- Rezeptor (BZR) Ag T-Zell- Rezeptor (TZR) MHC II MHC I Dendritische Zelle Ag-Prozessierung & Präsentation

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 ?)

Anatomical Location Lymph nodes Appendix Spleen Tonsils Peyer Plaques Afferent lymphatic vessel Paracortex (T cell zone) B cell zone medulla arteria Secundary B cell follicle „Germinal center“ Primary follicule Efferent venule Secondary lymphatic organs Lymph nodes Appendix Spleen Tonsils Peyer Plaques From Janeway

Long-lived plasma cells Anatomy of B Cell Response Spleen section - 7 days SRBC Antigen Expansion HEV IgM IgM T +/-TH T Cell Zone B Naive B cell +TH B cell focus Short-lived Plasma cell GC IgG IgA IgE Germinal center (GC) reaction: In response to T cell–dependent antigens and T cell help, B cells form GCs, where they undergo rapid proliferation, affinity maturation, and CSR. Memory cells and plasmablasts result from the GC reaction CXCR5 Secondary follicle + Germinal center Memory B cell Primary follicle B Cell Zone IgG IgA IgE IgD - B cells PNA - GC B cells CD3 - T cells Long-lived plasma cells

T-Zell-abhängige B-Zellaktivierung Lymphknoten Naive CD4-T CD8-T MHC II MHC I DZ TK T-Zellzone (extrafollikulär) Dendritische Zelle (DZ) TH Plasmazelle Gedächtnis-B B-Zellzone (follikulär) B TFH Infektion

T B Anatomy of B Cell Response GC T Cell Zone B Cell Zone Spleen section - 7 days SRBC Antigen Expansion HEV IgM IgM T +TH T Cell Zone B Naive B cell B cell focus Short-lived Plasma cell GC Germinal center (GC) reaction: In response to T cell–dependent antigens and T cell help, B cells form GCs, where they undergo rapid proliferation, affinity maturation, and CSR. Memory cells and plasmablasts result from the GC reaction Primary follicle B Cell Zone IgD - B cells PNA - GC B cells CD3 - T cells

Extrafollikuläre B/T-Zell-Kooperation B-Zell-Epitop (Peptid oder Hapten) Träger T-Zell-Epitop (Peptid) Gekoppelte Erkennung (Linked recognition) B- und T-Zellepitop müssen auf dem gleichen Molekül liegen IL2/4/5 ILR CD40 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 2 1 BZR CD40L + B MHC II Primed TH + Peptid TH TZR Immunologische Synapse

STUDON RINGVORLESUNG http://www.studon.uni-erlangen.de/crs816430.html

Long-lived plasma cells Anatomy of B Cell Response Spleen section - 7 days SRBC Antigen Expansion HEV IgM IgM T +/-TH T Cell Zone B Naive B cell +TH B cell focus Short-lived Plasma cell GC IgG IgA IgE Germinal center (GC) reaction: In response to T cell–dependent antigens and T cell help, B cells form GCs, where they undergo rapid proliferation, affinity maturation, and CSR. Memory cells and plasmablasts result from the GC reaction CXCR5 Secondary follicle + Germinal center Memory B cell Primary follicle B Cell Zone IgG IgA IgE IgD - B cells PNA - GC B cells CD3 - T cells 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

Germinal Center Reaction Modified from McHyzer-Williams 2011 B cell zone Light zone FDC TFH 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

Better and more specialized antibodies GC - Molecular Changes at the Ig locus IgM IgG, IgA, IgE VH AID VL CL AID tragets Cs in the V and the so-called switch regions upstream of the respective C region. The Us are removed and the gaps are repaied by error prone and mismatch repair. This results in point mutations in the V exon and strands breaks upstream of the C regions. The respective ends are ligated which CH Somatic hypermutation IgH class switch Better and more specialized antibodies

IgH Class Switch Recombination (CSR) Kinoshita & Honjo NRCB(2001)

IgH Class Switch Recombination (CSR) DNA-Looping-out und Deletion IL4 LPS VH Cm Cd Cg3 Cg1 Cg2b Cg2a Ce Ca IgM Sm Sg3 Sg1 Sg2b Sg2a Se Sa S, switch regions VH Cg1 Cg2b Cg2a Ce Ca Cd Cm Cg3 Cytokine AID IgG1 CSR VH Cg1 Cg2b Cg2a Ce Ca Switch circle Synapsis Incision Double-strand breaks End-Joining/Ligation Jäck et al.,P.N.A.S. USA 1988 von Schwedler et al., Nature 1990

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

Follicular dendritc cell (FDC) 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

Ag-specific antibodies 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

Germinal Center Reaction Modified from McHyzer-Williams 2011 B cell zone GC reaction Light zone FDC TFH Selection Selected B cell Dark zone Expansion SHM CSR? AID CSR TFH IgM T cell zone

B/T-Kooperation im Keimzentrum Nutt & Tarlinton, Nat Immunol. 2011

Germinal Center Reaction Modified from McHyzer-Williams 2011 B cell zone GC reaction Light zone FDC TFH 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

Control of PC Differentiation Staudt/ Calame/ Lassila Model PC Program Xbp1 Blimp1 IRF4↑↑↑↑ GC Program Pax5 AID IRF4 ↑ Repair Bcl6 Bach2 MiTF Modified from Nutt et al., 2011

Germinal Center Reaction Modified from McHyzer-Williams 2011 B cell zone GC reaction Light zone FDC TFH 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

Effector B Cells Plasma Cell (PC) Memory B Cell Igmem → Igsec IgG, IgE IgA Igmem → Igsec Ig production ↑ (100x) Long-lived PC in bone marrow niches Plasma Cell (PC) IgM IgM Plasma cell Ag 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 IgD IgG IgA IgE Naive B cell Memory B cell

Long-lived plasma cells Summary: Effector B Cells Clonal Expansion +/-TH Short-lived Plasma cells IgM +/-TH Differentiation IgM IgG, IgA, IgE 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

Adaptive Humoral Immunity AID Activation-Induced Deaminase Master regulator of secondary antibody diversification and ?????

AID - Entdeckung → Activation-Induced Deaminase = AID (Gensymbol AICD) 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 Deamidase R C O NH2 OH H2O NH3 Säureamid Carbonsäure AID Cytosin Uracil

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 5:51, 2008

APOBEC1 – Prototyp eines RNA-editierenden Enzyms Apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1 N C APOB100 Leber AUG CAA UAG APOB mRNA Duodenum APOBEC1 (one !!!! mutation in RNA) AUG UAA UAG Mut APOB mRNA Navaratnam et al., LBC 1993 Teng et al., Science, 1993 N C APOB48

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 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

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

AID – A Hypermutator → Activation-Induced Deaminase (AID) AID Cytosin 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

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)

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

RNA-Editing-Hypothese N C Aktiver „Mutator“ (Endonuklease) AID AUG CAA UAG „Mutator“encoding mRNA AID↑ in Keimzentrums-B-Zelle „Mutator“encoding mRNA AUG UAA UAG N C Inaktiver Mutator

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)

SHM CSR DNA Model Base excision and error prone Base excision and V exon C Cg C C CC C C CC U U UU U Base excision and error prone AID tragets Cs in the V and the so-called switch regions upstream of the respective C region. The Us are removed and the gaps are repaied by error prone and mismatch repair. This results in point mutations in the V exon and strands breaks upstream of the C regions. The respective ends are ligated which Base excision and mismatch repair A G A G

Three major DNA repairing mechanisms: 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

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. 

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.

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

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

In vitro, AID mutates ssDNA but not RNA 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

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?

AID – Zusammenfassung Activated B cell nucleus cytoplasm 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

Übersicht: Adaptive humorale Immunität Primärantwort Sekundärantwort IgM Kurzlebige Plasmazelle (IgM) Ag+TH IgM Kurzlebige Plasmazelle (IgM) Naive B-Zelle Ag+TH Naive B-Zelle Langlebige Plasmazelle (IgG, IgA, IgE) Gedächtnis- B-Zelle Keim- zentrums- reaktion Langlebige Plasmazelle (IgG, IgA, IgE) Gedächtnis- B-Zelle Keim- zentrums- reaktion Langlebige Plasmazelle (IgG, IgA, IgE) IgG 3 2 1 Schneller und mehr !!!! Log CAK im Serum IgM 10Ag 20Ag IgG IgM t (Tage) 0 4 8 0 4 8