DFG-Forschergruppe 832 Regulatoren der humoralen Immunantwort B Cell Club July 6, 2007 Function of IRF-4 in humoral immunity Hans-Martin Jäck Abteilung für Molekulare Immunologie an der Medizinische Klinik III

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 2 Interferon regulatory factor 4 (IRF-4) Member of the IRF transcriptin factor family Contains DNA binding domain with five conserved tryptophan repeats that interacts to regulatory elements in IFN-inducible genes Expression is limited to lymphoid (B and T) and myeloid lineages (DZ and MФ) Most closely related to IRF-8 in structure and expression pattern Functions in proliferation, apoptosis and differentiation Binding activity is modified by Ets (PU.1 and Spi-B) or helix-loop- helix (E2A) factors as well as Stat-6 and IRF-8 Biphasic expression in B lymphoid cells

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 3 Late Pro-B Late Pre-B Immature B Early Pre-B Mature B (+/- T H ) Primary Focus Secondary follicle Germinal center Mantel zone Primary follicle (+T H ) Centrocytes (CSR) Centroblasts (SHM) Memory B Secondary plasma blast Expression of IRF-4 in B Cell Lineage IRF-4 ↑ ↑

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 4 Antibody Repertoire Primary V H repertoire (specificity) Memory B cell Germinal Center  Somatic Hypermutation  Selection  IgH class switch  Memory B Plasma cell +T H IgG IgA IgE Modified secondary repertoire VH repertoire Increased affinity CH repertoire effector functions

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 5 Late Pro-B Late Pre-B Immature B cell Early Pre-B Stem cell Early Pro-B μHC fct ? LC ?  Igα KO  Igβ KO  µHC mem KO  μHC dys Pre-BCR: A Passport for Pre-B Cells to Multiply  IRF4/8 KO  Blink KO

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 6 Interferon regulatory factor 4 (IRF-4) Member of the IRF transcriptin factor family Contains DNA binding domain with five conserved tryptophan repeats that bind to regulatory elements in IFN-inducible genes Expression is limited to lymphoid and myeloid lineages (DZ and MФ) Most closely related to IRF-8 in structure and expression pattern Functions in proliferation, apoptosis and differentiation Binding activity is modified by Ets (PU.1 and Spi-B) or helix-loop- helix (E2A) factors as well as Stat-6 and IRF-8 Biphasic expression in B lymphoid cells Acts redundantly with IRF-8 in B cell maturation

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 7 Function of IRF-4 in humoral immunity Requirement of the Transcription Factor LSIRF/IRF4 in Mature B and T Lymphocytes (Mittrücker …….Mak, Science 27, 540, 1997) Graded Expression of Interferon Regulatory Factor-4 Coordinates Isotype Switching with Plasma Cell Differentiation (Sciammas …. Singh, Immunity 25, 225–236, 2006) Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination (Klein …. Dalla-Favera, NATURE IMMUNOLOGY 7, p773ff 2006)

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 8 IRF-4 deficient mouse (IRF-4-KO) Normal B and T cell maturation with normal numbers of peripheral B and T cell numbers Impairment at the transition of immature to mature B cell subsets

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 9 Low serum Ig levels (99% reduced) No germinal centers Fail to elicit T cell-dependent antigen-specific antibodies Reduced LPS-induced B cell proliferation Recall against DNP-KLH IRF-4 deficient mouse (IRF-4-KO)

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 10 No CD8 response against virus No tumor rejection IRF-4 deficient mouse (IRF-4-KO)

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 11 Interferon regulatory factor 4 (IRF-4) Member of the IRF transcriptin factor family Contains DNA binding domain with five conserved tryptophan repeats that bind to regulatory elements in IFN-inducible genes Expression is limited to lymphoid and myeloid lineages (DZ and MФ) Most closely related to IRF-8 in structure and expression pattern Functions in proliferation, apoptosis and differentiation Binding activity is modified by Ets (PU.1 and Spi-B) or helix-loop- helix (E2A) factors as well as Stat-6 and IRF-8 Biphasic expression in B lymphoid cells Acts redundantly with IRF-8 in B cell maturation Acts nonredundantly in B and T cell activation and generation of effector cells

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 12 Role of IRF-4 in post-GC B cell development C  1-Cre mouse eGFP IRF4 eGFP X Targeted IRF-4 mouse IRF-4 -/- and GFP + Immunisierung In vitro deletion with tat-cre (IRF-4 -/+ and GFP - ) Klein et al., 2006

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 13 Role of IRF-4 in post-GC B cell development Normal GC formation !!! Plasma cell generation requires IRF-4 IRF-4 deficient B cells fail to switch IgH isotype (low AID induction) Elispot 5d after immuniation for detection of IgG secreting plasma blast eGFP plus eGFP minus IgG1-secreting cells IRF4-minus IRF4-plus IgG1 GFP

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 14 IRF-4 may not be required for B memory formation since IgG1- positive GFP-positive B cells with somatic hypermutation could be detected IRF-4 not required for reactivation of memory B cells since recall increase IgG1- and GFP-positive B cells However, required for differentiation of memory B cells in plasma blasts, since IgG1-/GFP and CD138-postive cells were not detected after recall with NP-KLH Problem:You never know whether IRF4 had an effect before switch or whether small amounts of IRF4 are still present. Solution:Inducible IRF4 mouse IRF-4 and Memory B Cells

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 15 Mechanism by which IRF4 controls switchung and plasma cell differentiation IRF-4-KO mouse from Tak mak p-IRF4 IRF4 LPS Anti-CD40/IL4 GFP B cells IRF4- Retroviral transduction Proliferation Differentiation Expression of AID and Blimp1 Singh lab Immunity 2006

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 16 IRF-4 restores Ig secretion by upregulating Blimp1 IRF-4 transduction restores IgH switch by upregualtion AID IRF-4 controls swirch and Ig secretion IgG1 IRF4-GFP Control-GFP GFP

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 17 Problem: IRF-4 induces AID and Blimp1 program AID and Blimp1 are key components of antagonistic developmental stages

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 18 Solution: Graded expression Plasma blast (CD138) produce more IRF-4 than activated B cells Elevated abundance of IRF-4 in normal B cells upregulates Blimp-1 and down-regulates AID Blimp1 AID Transduced WT B cells after anti-CD40/IL4 stimulation

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 19

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 20 Late Pro-B Late Pre-B Immature B cell Early Pre-B Stem cell Early Pro-B μHC fct ? LC ? Mature B cell Vettermann et al. Sem. Immunol, 2006 D→ JHD→ JH V H →DJ H V L → J L B Cell Maturation and Ig Repertoire

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 21 Plasmazelle Naive, reife B-Zelle Ag (+/- T H ) IgM Primärer Fokus IgD IgM (in peripherären lymphatischen Organen) (+/- T H ) z.B. Lymphknoden Extrafollikulär Follikulär Sekundärer Follikel Keimzentrum Mantelzone Primärer Follikel (+T H )