A novel system to test for specificity of B cell receptors from tissue of Wegener's granulomatosis patients.
OBJECTIVE
Wegener's granulomatosis (WG) is characterized by granulomatous inflammation of the respiratory tract and Anti Neutrophil Cytoplasmic Antibody (ANCA)-associated systemic vasculitis. The pathognomonic ANCA in WG is typically directed against proteinase 3 (PR3). Germinal centre-like clusters of lymphocytes were seen in granulomata of WG patients suggesting an antigen-driven maturation of B lymphocytes potentially leading to ANCA formation. The goal of this study was to develop a system to determine the specificity of B cells found in WG granulomata via the generation of fab fragments as antibody analogues. These fab fragments have the identical antigen binding site like the B-cell receptor from which the DNA was derived.


METHODS
Single B cells were isolated from B cell clusters within the granuloma of a WG patient by laser-assisted microdissection. Their immunoglobulin genes (VH/Vkappa, VH/Vlambda) were characterized by seminested single cell PCR and cloned into a phagemid vector in order to produce fab fragments. The fabs were characterized by protein gel electrophoresis and western blot.


RESULTS
The immunoglobulin genes from lymphocyte infiltrates of WG granulomata reveal antigen-driven selection. On the basis of two individual couples of mutated VH/Vlambda PCR products functional fabs were generated that represent the B cell receptors of WG tissue-derived single B cells.


CONCLUSIONS
This is the first in vitro model to test for specificity of B cell receptors from WG granulomata. With respect to ANCA origin in WG this system provides a tool to elucidate the structure-function relationship of apparently antigen-driven maturation of B cells within Wegener's granuloma.
Voswinkel
J
J
Saarland University Medical School, Internal Medicine I, Homburg/Saar, Germany. injvos@uks.eu
Kerkdijk
A J
AJ
Mueller
A
A
Assmann
G
G
Pfreundschuh
M
M
Held
G
G
D019268:Antibodies, Antineutrophil Cytoplasmic / Q000276:immunology; D014890:Granulomatosis with Polyangiitis / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D066298:In Vitro Techniques; D011947:Receptors, Antigen, B-Cell / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D019268:Antibodies, Antineutrophil Cytoplasmic; D007140:Immunoglobulin Fab Fragments; D011947:Receptors, Antigen, B-Cell