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Kang Chen, Weifeng Xu, Melanie Wilson...Andrea Cerutti

ZIB Seminar, original report 12.10.2009 Asolina Braun. Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils. Kang Chen, Weifeng Xu, Melanie Wilson...Andrea Cerutti. Antibody structure. Vortrag.ppt 14.12.2006.

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Kang Chen, Weifeng Xu, Melanie Wilson...Andrea Cerutti

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  1. ZIB Seminar, original report 12.10.2009 Asolina Braun Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils Kang Chen, Weifeng Xu, Melanie Wilson...Andrea Cerutti

  2. Antibody structure Vortrag.ppt 14.12.2006

  3. Antibody isotypes

  4. Functions of different Ab isotypes Vortrag.ppt 14.12.2006

  5. Site of IgD production flow cytometry data • IgD+IgM- B cells are abundant in tonsil and nasal mucosa tissues • 0.5-1% IgD+IgM- of all CD19+ B cells in the blood • IgD+IgM- B cells showed a semi-differentiated plasma cell state (Blimp-1+, BCMA+ but CD138-; still Pax5+ & TACI+) • AID (activation-induced cytidine deaminase) expression suggests ongoing class switch recombination (CSR) tonsil tissue, 10x

  6. IgD class switch recombination • IgD switch occurs in respiratory mucosa Southern blot analysis of σδ-Sμ switch circles amplified by nested PCR from mononuclear cells of various tissues and hybridized with σδ or Sμ probes

  7. T-cell dependent follicular CSR: CD40 on B cells and CD40L on activated CD4+ T cells T-cell independent extrafollicular CSR: TACI & BAFF-R on B cells ligated by APRIL & BAFF (DC, MΦ, mucosal epithelial cells) test of combinations: CD40L/BAFF/APRIL alone or with IL-2, IL-4, IL-10, IL-13, IL-15, IL-21 isolation of IgD+IgM+ B cells => stimulation with ligand (and cytokine) for 4d/7d => southern blot to detect switch circles (4d), flow cytometry (7d), ELISA (7d) IgD class switch recombination

  8. IgD class switch recombination

  9. CD40L deficiency => HIGM1(hyper-IgM type1 syndrome) CD40 deficiency => HIGM3 AID deficiency => HIGM2 IgD CSR in immunodefficient patients => CD40 indispensable for systemic but not mucosal IgD

  10. isolation of circulating IgD+IgM+ B cells => stimulation 7d w/wo BAFF, IL-15, IL-21 => supernatant IgD for ELISA (n=5) IgD in respiratory mucosa MID = Moraxella catarrhalis IgD-binding protein LPS = Lipopolysaccharide CPS = capsular polysaccharide M. = Moraxella H. = Haemophilus

  11. polyclonal anti-IgD antibody used in flow cytometry Function of circulating IgD

  12. IgD intracellularly in circulating and mucosal basophils binding capacity of IgD on basophils is saturated binding of IgD to pre-basophilic cell line KU812, mastocytoid HMC-1 & LAD-2, monocytic U937, but not to T cell, NK cell or myeloid cell lines KU812 and LAD-2 bound more IgD after stimulation with IL-3 and/or IL-4 (and upregulated IL-4 R, IL-3 R, IgE R) Mannose and mannan slightly inhibited IgD binding to HMC-1 IgD binding was abolished by denaturation of IgD or pretreatment of cells with trypsin or papain IgD binding on basophils and mast cells depends on a protease sensitive receptor that is IL-3 and IL-4 inducible IgD on basophils

  13. basophils are known to bind IgE, crosslinking of bound IgE leads to degranulation and histamin release isolation of basophils => stimulation with beads or Ig-coated beads => 30min/5h => flow cytometry => granular CD63 as a marker for activation Implications of IgD binding

  14. incubation of basophils with microbeads w/wo IgE/IgD & w/wo Il-3 => 16h/48h => ELISA Implications of IgD binding IL-4: B cell activation, IgE switch IL-13: B cell growth & differentiation BAFF: B cell proliferation IL-8: chemokine for basophils, neutrophils, T cells CXCL10: chemokine for monocytes, NK cells, naive T cells

  15. IgD-stimulated basophils produce antimicrobial, opsonizing and alarm-signaling factors (ß-defensins,cathelicidin, CRP, SPAG11, PTX3) supernatant of activated basophils (IgD beads) inhibits bacterial replication: Implications of IgD binding

  16. IgD class switch recombination (CSR) in respiratory mucosa produces IgD+IgM- B cells IgD CSR depends on CD40L, BAFF, APRIL and needs IL-2, IL-15 and IL-21 mucosal IgD recognizes respiratory bacteria secreted IgD binds to basophils and mast cells IgD binding to basophils does not elicit degranulation or histamine release, but upregulates IL-4, IL13, BAFF, IL-8, CXL10 (B cell activation) produce antimicrobial, opsonizing and alarm-signaling factors inhibit bacterial replication Summary

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