Leyendeckers Heike, Tasanen Kaisa, Bruckner-Tuderman Leena, Zillikens Detlef, Sitaru Cassian, Schmitz Jürgen, Hunzelmann Nicolas
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
J Invest Dermatol. 2003 Mar;120(3):372-8. doi: 10.1046/j.1523-1747.2003.12071.x.
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.
大疱性类天疱疮是一种表皮下疱病，其特征是合成针对180 kDa和230 kDa大疱性类天疱疮抗原的自身抗体。循环中自身抗原特异性记忆B细胞的存在是否也反映自身免疫仍存在争议。我们使用一种新的检测方法，将两步免疫磁珠富集与多参数流式细胞术相结合，以检测和表征大疱性类天疱疮患者血液中针对大疱性类天疱疮180 kDa的特异性IgG⁺B细胞。在第一次磁珠分离中，使用与CD19抗体偶联的可释放微珠从外周血单核细胞中分离出B细胞。然后，使用与大疱性类天疱疮180 kDa胞外域的重组N端片段直接偶联的微珠，从预富集的B细胞中阳性选择大疱性类天疱疮180 kDa特异性细胞，该片段包含非胶原16A结构域，最近研究表明该结构域包含大疱性类天疱疮血清中自身抗体的主要表位。在大多数（如果不是全部）血清中存在针对非胶原16A结构域自身抗体的患者血液中，可检测到非胶原16A结构域特异性IgG⁺B细胞。通过体外分化为抗体形成细胞并分析培养上清液中是否存在非胶原16A结构域特异性IgG抗体，证实了这些细胞的特异性。所有非胶原16A结构域特异性IgG⁺B细胞均表现出明确的记忆免疫表型。非胶原16A结构域特异性IgG⁺记忆B细胞可能对于持续产生非胶原16A结构域特异性自身抗体至关重要，和/或作为抗原呈递细胞，在启动和再刺激大疱性类天疱疮180 kDa特异性辅助性T细胞中发挥作用。
