1. Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
2. ^*Department of Dermatology, University of Oulu, Oulu, Finland
3. ^†Department of Dermatology, University of Münster, Münster, Germany
4. ^‡Department of Dermatology, University of Würzburg, Würzburg, Germany
5. ^§Department of Dermatology, University of Cologne, Cologne, Germany

Correspondence: Nicolas Hunzelmann, MD, Klinik für Dermatologie, Universität zu Köln, Josef-Stelzmann-Str. 9, D-50931 Köln, Germany. Email: Nico.Hunzelmann@uni-koeln.de

Received 23 April 2002; Revised 27 September 2002; Accepted 18 October 2002.

Abstract

Bullous pemphigoid is a subepidermal blistering disease characterized by the synthesis of autoantibodies against the 180 kDa and the 230 kDa bullous pemphigoid antigens. Whether autoimmunity is also reflected by the presence of circulating autoantigen-specific memory B cells is still a matter of debate. We used a new assay combining two-step immunomagnetic enrichment with multiparameter flow cytometry to detect and characterize bullous pemphigoid 180 kDa-specific IgG⁺ B cells in blood of bullous pemphigoid patients. In a first magnetic separation, B cells were isolated from peripheral blood mononuclear cells using releasable microbeads conjugated to a CD19 antibody. From pre-enriched B cells, bullous pemphigoid 180 kDa-specific cells were then positively selected using microbeads directly conjugated with a recombinant N-terminal fragment of the bullous pemphigoid 180 kDa ectodomain, containing the noncollagenous 16A domain, which was recently shown to harbor major epitopes of autoantibodies in bullous pemphigoid sera. Noncollagenous 16A domain-specific IgG⁺ B cells were detectable in blood of most, if not all patients with serum autoantibodies against the noncollagenous 16A domain. The specificity of the cells was confirmed by in vitro differentiation into antibody-forming cells and analysis of the culture supernatant for the presence of noncollagenous 16A domain-specific IgG antibodies. All noncollagenous 16A domain-specific IgG⁺ B cells showed a clear memory immunophenotype. Noncollagenous 16A domain-specific IgG⁺ memory B cells may be crucial for continuous noncollagenous 16A domain-specific autoantibody production and/or play a part as antigen-presenting cells for priming and restimulation of bullous pemphigoid 180 kDa-specific T helper cells.