Hans-Martin Jäck Abteilung für Molekulare Immunologie Medizinische Klinik III

1. 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

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

7. VH CH1 VL CL L CH2 CHO H CH3 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

8. 3 loops (fingers) from each V region form the antigen binding site (paratop) CDRs = complementary determining regions 1-3 VH 3 2 VL 1 L 3 2 • Tissue distribution • Serum halflife • Complement • Phagocytosis • Recruting of cells 1 H 1 Effector sites “Bullet Part” 2 3 Janeway Antibodies – Structure and Function Antigen binding sites = Paratop “Magic Part” VH VL CH CL CH CH CH Antibodies are bifunctional (Paul Ehrlich‘s 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) BCR Pre-BCR Stem cell Late Pro-B Early Pre-B Late Pre-B Immature B cell VH→D →JH VL →JL L H

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

13. 109-1012 anti- bodies ca. 107 anti- bodies Summary: Preimmune Repertoire 4JH 134 VH C 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

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

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

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

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

18. 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 B cell help in follicule

19. Differentiation Clonal Expansion IgM Short-lived Plasma cells +/-TH +/-TH IgG, IgA, IgE +TH Long-lived plasma cells +TH Memory B cell B Cell Antigens The World of Antigens (Antibodygenerating) • 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

20. Ag B-Zell- Rezeptor (BZR) MHC I MHC II T-Zell- Rezeptor (TZR) 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. B cell zone Primary B cell follicule medulla arteria Paracortex (T cell zone) venule Afferent lymphatic vessel Efferent lymphatic vessel Secundary B cell follicle „Germinal center“ Anatomical Location Secondary lymphatic organs Lymphnodes Appendix Spleen Tonsils PeyerPlaques From Janeway

23. Primary follicle Secondary follicle + Germinal center Anatomyof B Cell Response Spleen section - 7 days SRBC Antigen Expansion HEV IgM IgM T +/-TH T Cell Zone B Naive B cell Short-lived Plasma cell +TH B cell focus 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

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

25. Primary follicle Anatomyof B Cell Response Spleen section - 7 days SRBC Antigen Expansion HEV IgM 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

26. 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 Träger CD40L Immunologische Synapse 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 2 BZR + B MHC II Primed TH + Peptid TH TZR

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

28. Primary follicle Secondary follicle + Germinal center Anatomyof B Cell Response Spleen section - 7 days SRBC Antigen Expansion HEV IgM IgM T +/-TH T Cell Zone B Naive B cell Short-lived Plasma cell +TH B cell focus 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 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 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 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

31. GC - MolecularChangesattheIglocus IgM IgG, IgA, IgE VH AID VL CL CH • Somatic hypermutation • IgH class switch Betterandmorespecializedantibodies

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

33. CSR IgG1 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 Cd AID Cg3 Cm VH Cg1 Cg2b Cg2a Ce Ca • Synapsis • Incision • Double-strand breaks • End-Joining/Ligation Switch circle VH Cg1 Cg2b Cg2a Ce Ca 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 couldbeselectedbyself-antigen on FDC – ► Requirementforanothercheckpoint

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

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

41. Germinal Center Reaction Modified from McHyzer-Williams 2011 B cell zone GCreaction 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

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

43. Differentiation Clonal Expansion IgM Short-lived Plasma cells +/-TH +/-TH Summary: Effector B Cells • Better and more • specialized abs IgM 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 IgG, IgA, IgE +TFH Memory B cell AID

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

45. Deamidase AID Uracil Cytosin H2O NH3 O O R C R C NH2 OH Säureamid Carbonsäure 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 5:51, 2008

47. APOBEC1 (one !!!! mutation in RNA) Duodenum 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 gegenViren 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 Cytosin Uracil 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