Determination of human γδ T cell-mediated cytotoxicity using a non-radioactive assay system

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 [⁵¹Cr]-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 (Eu³⁺) 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 Eu³⁺ and terbium (Tb³⁺) 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 Eu³⁺ and Tb³⁺, Tb³⁺ chelate was more sensitive than Eu³⁺ chelate, suggesting that the detection system using Tb³⁺ is superior to Eu³⁺ 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.