免疫缺陷小鼠体内人源T淋巴细胞亚群流式检测方法研究

Study on flow cytometry method of human T lymphocyte subpopulations in immunodeficient mice

目的比较流式细胞术(FCM)与活体成像法、qPCR法识别外周血CAR^(+)细胞的结果,验证其结果的可靠性和灵敏性。方法分别给予免疫缺陷型荷瘤(Raji-Luc细胞造模)小鼠溶媒、CAR-T1或CAR-T2细胞。在不同时间点测定动物体内Raji-Luc细胞的荧光强度,FCM法测定人源T淋巴细胞和CAR-T细胞中的CD3^(+)CD4^(+)和CD3^(+)CD8^(+)个数,并使用qPCR法检测外周血中每纳克基因组DNA中CAR-T1和CAR-T2拷贝数。结果CAR-T1组动物体内T细胞及CAR-T细胞数量在给予细胞后第5~7周大幅增加。CAR-T2组动物体内T细胞及CAR-T细胞数量在给予细胞后第10~14天大幅增加,之后大幅降低,给予细胞后第4周降至基线且未见再次扩增。CAR-T细胞的流式检测结果与活体成像结果基本相符,且与qPCR法检测外周血中CAR-T细胞的分布趋势基本一致。此外,EDTA-K2抗凝剂对FITC标记的抗CD8^(+)抗体有较明显的影响,导致标记为CD3^(+)CD8^(+)的细胞群左移且检测结果为零。结论对不同增殖特性的CAR-T细胞,流式检测结果与其体内肿瘤杀伤情况相关,且比qPCR法更为准确和灵敏,为CAR-T细胞的非临床安全性评价试验设计提供有益借鉴。

Objective To compare the results of flow cytometry(FCM), in vivo imaging and quantitative polymerase chain reaction(qPCR) to identify CAR^(+) cells in peripheral blood, and to verify the reliability and sensitivity of the results.Methods Immunodeficient tumor-bearing(Raji-Luc cell model) mice were given vehicle, CAR-T1 or CAR-T2 cells, respectively.The fluorescence intensities of Raji-Luc cells in animals were measured at different time points, the number of CD3^(+)CD4^(+) and CD3^(+)CD8^(+) in human T lymphocytes and CAR-T cells were determined by FCM method, and the copy numbers of CAR-T1 and CAR-T2 per ng genomic DNA in peripheral blood were detected by qPCR method.Results The number of T cells and CAR-T cells in the animals in the CAR-T1 group increased significantly from 5 to 7 weeks after the cells were administered. The numbers of T cells and CAR-T cells in the animals in the CAR-T2 group increased significantly from 10 to 14 days after administration of cells, and then decreased significantly, and dropped to baseline at 4 weeks after administration of cells without re-expansion. The flow detection results of CAR-T cells were consistent with the in vivo imaging results, and were basically consistent with the distribution trend of CAR-T cells in peripheral blood detected by qPCR. In addition,EDTA-K2 anticoagulant had a significant effect on the FITC-labeled anti-CD8^(+) antibody, and resulted in a left shift of the cell population labeled as CD3^(+)CD8^(+), with a detection result of zero. After labeling, the CD3^(+)CD8^(+) cells shifted to the left until the detection of CD3^(+)CD8^(+) was zero. Conclusion This study has compared CAR-T cells with different proliferation characteristics, and verified that the results of flow cytometry are correlated with their in vivo tumor killing, and the results are more accurate and sensitive than qPCR. The research results provide a useful reference for the design of non-clinical safety evaluation trials of CAR-T cells.