Center for Immune Regulation, Oslo University Hospital, Oslo, Norway.
J Immunol. 2010 Nov 1;185(9):5377-83. doi: 10.4049/jimmunol.1001587. Epub 2010 Oct 8.
The gut mucosal surface is efficiently protected by Abs, and this site represents one of the richest compartments of Ab-secreting cells in the body. A simple and effective method to generate Ag-specific human monoclonal Abs (hmAbs) from such cells is lacking. In this paper, we describe a method to generate hmAbs from single Ag-specific IgA- or IgM-secreting cells of the intestinal mucosa. We found that CD138-positive plasma cells from the duodenum expressed surface IgA or IgM. Using eGFP-labeled virus-like particles, we harnessed the surface Ig expression to detect rotavirus-specific plasma cells at low frequency (0.03-0.35%) in 9 of 10 adult subjects. Single cells were isolated by FACS, and as they were viable, further testing of secreted Abs by ELISPOT and ELISA indicated a highly specific selection procedure. Ab genes from single cells of three donors were cloned, sequenced, and expressed as recombinant hmAbs. Of 26 cloned H chain Ab genes, 22 were IgA and 4 were IgM. The genes were highly mutated, and there was an overrepresentation of the VH4 family. Of 10 expressed hmAbs, 8 were rotavirus-reactive (6 with K(d) < 1 × 10(-10)). Importantly, our method allows generation of hmAbs from cells implicated in the protection of mucosal surfaces, and it can potentially be used in passive vaccination efforts and for discovery of epitopes directly relevant to human immunity.
肠道黏膜表面被 Abs 有效地保护着，而这个部位是体内分泌 Ab 细胞最丰富的部位之一。目前还缺乏一种从这些细胞中生成抗原特异性人源单克隆 Abs（hmAbs）的简单而有效的方法。在本文中，我们描述了一种从肠道黏膜中抗原特异性 IgA 或 IgM 分泌细胞中生成 hmAbs 的方法。我们发现，十二指肠的 CD138 阳性浆细胞表达表面 IgA 或 IgM。利用 eGFP 标记的病毒样颗粒，我们利用表面 Ig 表达，在 10 名成人受试者中的 9 名中以低频率（0.03-0.35%）检测到轮状病毒特异性浆细胞。通过 FACS 分离单细胞，由于它们具有活力，通过 ELISPOT 和 ELISA 进一步检测分泌的 Abs 表明这是一种高度特异性的选择程序。从 3 名供体的单个细胞中克隆、测序并表达了 Ab 基因，得到了重组 hmAbs。在 26 个克隆的 H 链 Ab 基因中，22 个是 IgA，4 个是 IgM。这些基因高度突变，VH4 家族的表达过剩。在 10 个表达的 hmAbs 中，有 8 个对轮状病毒有反应（6 个 K(d) < 1×10(-10)）。重要的是，我们的方法允许从参与保护黏膜表面的细胞中生成 hmAbs，并且它可以潜在地用于被动免疫接种努力和发现与人类免疫直接相关的表位。
