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 Antibody isotypes Functions of different Ab isotypes Vortrag.ppt 14.12.2006 Site of IgD production • 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 semidifferentiated plasma cell state (Blimp-1+, BCMA+ but CD138-; still Pax5+ & TACI+) flow cytometry data • AID (activation-induced cytidine deaminase) expression suggests ongoing class switch recombination (CSR) tonsil tissue, 10x 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 IgD class switch recombination • 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 IgD CSR in immunodefficient patients • CD40L deficiency => HIGM1(hyper-IgM type1 syndrome) CD40 deficiency => HIGM3 AID deficiency => HIGM2 => CD40 indispensable for systemic but not mucosal IgD IgD in respiratory mucosa • isolation of circulating IgD+IgM+ B cells => stimulation 7d w/wo BAFF, IL-15, IL-21 => supernatant IgD for ELISA (n=5) MID = Moraxella catarrhalis IgD-binding protein LPS = Lipopolysaccharide CPS = capsular polysaccharide M. = Moraxella H. = Haemophilus Function of circulating IgD • polyclonal anti-IgD antibody used in flow cytometry IgD on basophils • 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 Implications of IgD binding • • 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 • incubation of basophils with microbeads w/wo IgE/IgD & w/wo Il-3 => 16h/48h => ELISA 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 Implications of IgD binding • IgD-stimulated basophils produce antimicrobial, opsonizing and alarmsignaling factors (ß-defensins,cathelicidin, CRP, SPAG11, PTX3) • supernatant of activated basophils (IgD beads) inhibits bacterial replication: Summary • 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