A label-free and real-time method for evaluating cytotoxicity of CD19 CAR-T cells / 医学研究生学报

ABSTRACT

ObjectiveUsing Chromium-51 release assay, lactate dehydrogenase release assayand other methods to detect the cytotoxicity of CD19 CAR-T cells is cumbersome, with low repeatability and poor stability. This study aims to establish a label-free and real-time method for detectingspecific cytotoxicity of CD19 CAR-T cells.MethodsIn order to establish target cell models for cytotoxic assay of CD19 CAR-T cells by using Real Time Cellular Analysis (RTCA) system,the adherent human breast cancer cells were infected with lentiviral vectors encoding CD19. CD19 expression on the transduced cells was detected by flow cytometry. The cellsexpressing CD19 stably werethen sorted by fluorescence activated cell sorting (FACS).With such cells as target cells, CD19 CAR-T cells and BCMA CAR-T cells as effector cells, RTCAsystem was used to evaluate the cytotoxicity of CAR-T cells against target cells.ResultsMDA-MB-231 and SKBR3cells with stable expression CD19were obtained in this study.The results of flow cytometry showed that positive expression rate of CD19 in MDA-MB-231/CD19 cells and SKBR3/CD19 monoclonal cells were 99.03% and 98.91%,respectively.RTCA results showed that with MDA-MB-231 and MDA-MB-231/CD19 cells as target cells,CD19 CAR-T cells showed significant cytotoxicity to MDA-MB-231/CD19 cellsat the effector-target ratio of 5∶1, 1∶1 and 1∶5,but not to MDA-MB-231 cells. With SKBR3 and SKBR3/CD19 cells as target cells, CD19 CAR-T cells showed significant cytotoxicity to SKBR3/CD19 cellsat the effector-target ratio of 5∶1and 1∶1. When the effector-target ratio was 1∶5, there was no obvious cytotoxicity.The data of MDA-MB-231/CD19 or SKBR3/CD19 as target cells and CD19 CAR-T as effector cells were analyzed separately, showing that when the number of target cells was the same, the cytotoxicity detected by RTCA increased as the number of CD19 CAR-T cells increased.The cytotoxic assays of CD19 CAR-T cells showed specificity and dose-response relationship of CD19 CAR-T cytotoxicity against the target cells.ConclusionThis study established a method for evaluating cytotoxicity of CD19 CAR-T cells that is real-time, label-free, simple and convenient.