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

Antibody structure

Vortrag.ppt14.12.2006

Antibody isotypes

Functions of different Ab isotypes

Vortrag.ppt14.12.2006

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 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

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 => HIGM3AID 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 proteinLPS = LipopolysaccharideCPS = capsular polysaccharide

M. = MoraxellaH. = 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 switchIL-13: B cell growth & differentiationBAFF: B cell proliferationIL-8: chemokine for basophils, neutrophils, T cellsCXCL10: chemokine for monocytes, NK cells, naive T cells

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:

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