Department of Biomedicine, Aarhus University, Aarhus C, Denmark.
Clin Exp Immunol. 2013 Sep;173(3):398-410. doi: 10.1111/cei.12133.
Damage of target cells by cytotoxicity, either mediated by specific lymphocytes or via antibody-dependent reactions, may play a decisive role in causing the central nervous system (CNS) lesions seen in multiple sclerosis (MS). Relevant epitopes, antibodies towards these epitopes and a reliable assay are all mandatory parts in detection and evaluation of the pertinence of such cytotoxicity reactions. We have adapted a flow cytometry assay detecting CD107a expression on the surface of cytotoxic effector cells to be applicable for analyses of the effect on target cells from MS patients expressing increased amounts of human endogenous retrovirus antigens. MS patients also have increased antibody levels to these antigens. The target cells are spontaneously growing peripheral blood mononuclear cells (PBMCs) of B cell lineage, expressing human endogenous retrovirus HERV epitopes on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used as control antibody. Without antibodies this system is suitable for analyses of natural killer cell activity. In optimization of the assay we have used effector lymphocytes from healthy donors. The most effective effector cells are CD56(+) cells. CD8(+) T cells also express CD107a in ADCC. Using the adapted assay, we demonstrate significant ADCC activity to target cells expressing HERV epitopes, and additionally a low level of NK activity.
细胞毒性引起的靶细胞损伤，无论是由特异性淋巴细胞介导还是通过抗体依赖性反应引起的，可能在多发性硬化症（MS）中中枢神经系统（CNS）病变的发生中起决定性作用。相关表位、针对这些表位的抗体和可靠的检测方法都是检测和评估这种细胞毒性反应相关性的必要组成部分。我们已经适应了一种流式细胞术检测细胞毒性效应细胞表面 CD107a 表达的方法，以适用于分析表达人内源性逆转录病毒抗原的 MS 患者靶细胞的影响。MS 患者对这些抗原的抗体水平也升高了。靶细胞是自发生长的外周血单核细胞（PBMC），其表面表达人类内源性逆转录病毒 HERV 表位。针对 HERVs 的Env 和 gag 区域中定义的肽的多克隆抗体在兔子中产生，并用于抗体依赖性细胞介导的细胞毒性（ADCC）测定中。利妥昔单抗®（罗氏）是一种针对主要表达于 B 细胞上的 CD20 的嵌合单克隆抗体，用作对照抗体。在没有抗体的情况下，该系统适用于自然杀伤细胞活性的分析。在优化测定法时，我们使用了来自健康供体的效应淋巴细胞。最有效的效应细胞是 CD56（+）细胞。CD8（+）T 细胞在 ADCC 中也表达 CD107a。使用适应的测定法，我们证明了针对表达 HERV 表位的靶细胞具有显著的 ADCC 活性，并且还具有低水平的 NK 活性。
