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DFG-Forschergruppe 832 Regulatoren der humoralen Immunantwort

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. Interferon regulatory factor 4 (IRF-4).

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DFG-Forschergruppe 832 Regulatoren der humoralen Immunantwort

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  1. 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

  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

  3. Expression of IRF-4 in B Cell Lineage (+/- TH) Primary Focus IRF-4 ↑ ↑ Late Pro-B Late Pre-B ImmatureB Mature B Early Pre-B IRF-4 ↑ ↑ (+TH) IRF-4 ↑ ↑ Memory B Centrocytes (CSR) Centroblasts (SHM) Secondary plasma blast Mantel zone Germinal center Primary follicle Secondary follicle IRF-4 ↑ ↑

  4. IRF4/8 KO • Blink KO • Igα KO • Igβ KO • µHCmem KO • μHCdys Pre-BCR: A Passport for Pre-B Cells to Multiply μHCfct? LC ? Stem cell Early Pro-B Late Pro-B Late Pre-B ImmatureB cell Early Pre-B

  5. 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

  6. 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)

  7. 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

  8. IRF-4 deficient mouse (IRF-4-KO) • 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

  9. IRF-4 deficient mouse (IRF-4-KO) • No CD8 response against virus • No tumor rejection

  10. 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

  11. Role of IRF-4 in post-GC B cell development Cg1-Cre mouse X Targeted IRF-4 mouse (IRF-4 -/+and GFP-) IRF4 eGFP In vitro deletion with tat-cre Immunisierung eGFP IRF-4 -/- and GFP+ Klein et al., 2006

  12. eGFP minus eGFP plus IgG1-secreting cells Elispot 5d after immuniation for detection of IgG secreting plasma blast 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) IRF4-minus IgG1 IRF4-plus GFP

  13. IRF-4 and Memory B Cells • 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

  14. Mechanism by which IRF4 controls switchung and plasma cell differentiation IRF-4-KO mouse from Tak mak LPS Anti-CD40/IL4 • Proliferation • Differentiation • Expression of AID • and Blimp1 IRF4- GFP B cells p-IRF4 Singh lab Immunity 2006 IRF4 Retroviral transduction

  15. IRF4-GFP Control-GFP IgG1 GFP IRF-4 controls swirch and Ig secretion • IRF-4 restores Ig secretion by upregulating Blimp1 • IRF-4 transduction restores IgH switch by upregualtion AID

  16. Problem: IRF-4 induces AID and Blimp1 program AID and Blimp1 are key components of antagonistic developmental stages

  17. Blimp1 AID Transduced WT B cells after anti-CD40/IL4 stimulation 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

  18. D→JH VH →DJH VL →JL B Cell Maturation and Ig Repertoire μHCfct? LC ? Stem cell Early Pro-B Late Pro-B Early Pre-B Late Pre-B ImmatureB cell Mature B cell Vettermann et al. Sem. Immunol, 2006

  19. IgD Sekundärer Follikel PrimärerFollikel (+TH) Keimzentrum Mantelzone z.B. Lymphknoden Ag Primärer Fokus Plasmazelle IgM Extrafollikulär IgM (+/- TH) (+/- TH) Naive, reife B-Zelle Follikulär (in peripherären lymphatischen Organen)

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