Wardemann Hedda, Busse Christian E
Division of B-Cell Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Methods Mol Biol. 2019;1956:105-125. doi: 10.1007/978-1-4939-9151-8_5.
The majority of lymphomas originate from B cells at the germinal center stage. Preferential selection of B-cell clones by a limited set of antigens has been suggested to drive lymphoma development. While recent studies in chronic lymphocytic leukemia have shown that self-reactive B-cell receptors (BCR) can generate cell-autonomous signaling and proliferation, our knowledge about the role of BCRs for the development or survival of other lymphomas remains limited. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire at single-cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow-cytometric isolation of single human B cells to the reverse transcription-polymerase chain reaction (RT-PCR)-based amplification of the expressed immunoglobulin (Ig) transcripts (IGH, IGK, and IGL) and their subsequent cloning into expression vectors for the in vitro production of recombinant monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B-cell lymphomas by single-cell sequencing of Ig transcripts and on the antibody reactivity of human lymphoma B cells.
大多数淋巴瘤起源于生发中心阶段的B细胞。有限的一组抗原对B细胞克隆的优先选择被认为是驱动淋巴瘤发展的原因。虽然最近对慢性淋巴细胞白血病的研究表明，自身反应性B细胞受体（BCR）可产生细胞自主信号和增殖，但我们对BCR在其他淋巴瘤发生或存活中的作用的了解仍然有限。在此，我们描述了一种表征人B细胞抗体反应性的策略。该方法通过从特定来源的单个原代人B细胞生成重组单克隆抗体，能够在单细胞水平上对人抗体库进行无偏倚的表征。本方案详细描述了该方法，从流式细胞术分离单个B细胞开始，到基于逆转录-聚合酶链反应（RT-PCR）扩增表达的免疫球蛋白（Ig）转录本（IGH、IGK和IGL），以及随后将它们克隆到表达载体中以体外生产重组单克隆抗体。该策略可用于通过Ig转录本的单细胞测序获得有关B细胞淋巴瘤克隆进化的信息，以及有关人淋巴瘤B细胞抗体反应性的信息。
