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Immunoglobulin D enhances immune surveillance
by activating antimicrobial, proinflammatory and
B cell-stimulating programs in basophils
Kang Chen, Weifeng Xu, Melanie Wilson...Andrea Cerutti
ZIB Seminar, original report 12.10.2009 Asolina Braun
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Antibody structure
Vortrag.ppt14.12.2006
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Antibody isotypes
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Functions of different Ab isotypes
Vortrag.ppt14.12.2006
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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 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)
flow cytometry data
tonsil tissue, 10x
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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
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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)
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IgD class switch recombination
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IgD CSR in immunodefficient patients• CD40L deficiency => HIGM1(hyper-IgM type1 syndrome)
CD40 deficiency => HIGM3AID deficiency => HIGM2
=> CD40 indispensable for systemic but not mucosal IgD
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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 polysaccharideM. = MoraxellaH. = Haemophilus
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Function of circulating IgD
• polyclonal anti-IgD antibody used in flow cytometry
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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
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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
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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 switchIL-13: B cell growth & differentiationBAFF: B cell proliferationIL-8: chemokine for basophils, neutrophils, T cellsCXCL10: chemokine for monocytes, NK cells, naive T cells
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Implications of IgD binding• 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:
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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