A novel system to test for specificity of B cell receptors from tissue of Wegener`s granulomatosis patients

CER3317
2008 Vol.26, N°3 ,Suppl.49
PI 0090, PF 0096
Full Papers

Free to view
(click on article PDF icon to read the article)

PMID: 18799062 [PubMed]

Abstract

OBJECTIVES:
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/VΚ, VH/V&lgr;) 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/V&lgr; 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.