Center for Bioinformatics and Molecular Medicine, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; Program for Nurturing Global Leaders in Tropical and Emerging Infectious Diseases, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
Center for Bioinformatics and Molecular Medicine, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
J Immunol Methods. 2019 Mar;466:32-40. doi: 10.1016/j.jim.2019.01.003. Epub 2019 Jan 14.
The adoptive transfer of immune effector cells, such as CD8 killer αβ T cells, γδ T cells, NK (natural killer) cells, and genetically-modified T cells, has been receiving increasing attention. It is essential to determine cellular cytotoxicity so as to monitor the function and quality of ex vivo-expanded immune effector cells before infusion. The most common method is the [Cr]-sodium chromate release assay. It is, however, preferable to avoid the use of radioactive materials in clinical laboratories. In order to establish a non-radioactive alternative to the standard radioactive assay, we previously synthesized a chelate-forming prodrug (BM-HT) and demonstrated that a combination of BM-HT and europium (Eu) was useful to determine NK cell-mediated cytotoxicity. In the present study, we examined whether or not this improved assay system could be used to determine the cellular cytotoxicity exhibited by Vγ2Vδ2 γδ T cells. In addition, we compared Eu and terbium (Tb) in the measurement of cellular cytotoxicity. Our assay system using BM-HT could be used successfully for the analysis of both γδ T cell receptor (TCR)- and CD16-mediated cytotoxicity. When the intensity of fluorescence was compared between Eu and Tb, Tb chelate was more sensitive than Eu chelate, suggesting that the detection system using Tb is superior to Eu when tumor cells are not efficiently labeled with BM-HT. The method established herein is expected to promote the development of novel adoptive cell therapies for cancer.
免疫效应细胞的过继转移，如 CD8 杀伤性 αβ T 细胞、γδ T 细胞、NK（自然杀伤）细胞和基因修饰的 T 细胞，正受到越来越多的关注。在输注前，确定细胞毒性对于监测体外扩增的免疫效应细胞的功能和质量至关重要。最常用的方法是 [Cr]- 酸钠放射性铬释放试验。然而，在临床实验室中最好避免使用放射性物质。为了建立一种非放射性的替代标准放射性检测方法，我们之前合成了一种形成螯合物的前体药物（BM-HT），并证明 BM-HT 与铕（Eu）的组合可用于测定 NK 细胞介导的细胞毒性。在本研究中，我们研究了该改进的检测系统是否可用于测定 Vγ2Vδ2 γδ T 细胞的细胞毒性。此外，我们比较了 Eu 和铽（Tb）在细胞毒性测量中的应用。我们使用 BM-HT 的检测系统可成功用于分析 γδ TCR 和 CD16 介导的细胞毒性。当比较 Eu 和 Tb 的荧光强度时，Tb 螯合物比 Eu 螯合物更敏感，这表明当肿瘤细胞不能有效地被 BM-HT 标记时，使用 Tb 的检测系统优于 Eu。本文建立的方法有望促进癌症的新型过继细胞疗法的发展。
