Ren C X, Chen X X, Zhao L, Tian Y, Xu K L, Zhao K
Blood Disease Institute, Xuzhou Medical University, Key Laboratory of Bone Marrow Stem Cell, Department of Hematology, the Affiliated Hospital of Xuzhou Medical University, Xuzhou 221000, China.
Zhonghua Xue Ye Xue Za Zhi. 2022 Jun 14;43(6):506-512. doi: 10.3760/cma.j.issn.0253-2727.2022.06.011.
To optimize the stimulation and activation system of mouse CD3(+) T cells in vitro and explore the optimal infection time of CD3(+) T cells to establish mouse CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and to also verify its killing effect in vivo and in vitro. Splenic CD3(+)T cells were isolated and purified using magnetic beads, and the cells were cultured in Soluble anti-CD3/CD28, PMA+Ionomycin, and Plated anti-CD3/CD28. Cell activation and apoptosis were assessed by flow cytometry after 8, 24, 48, and 72 hours. ScFv plasmid of mouse CD19 antibody was transfected to plat-E cells to package retrovirus. Activated CD3(+) T cells were infected to construct mouse-specific CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and mCD19 CAR-T cells were co-cultured with B-cell lymphoma cell line A20 in vitro. The specific toxicity of A20 was detected by flow cytometry, and mCD19 CAR-T cells were infused into the lymphoma mouse model to detect its killing effect and distribution. The activation effect of Plated anti-CD3/CD28 on CD3(+) T cells was superior, with the cells exhibiting good viability 24-48 hours after stimulation. Established mCD19 CAR-T cells with stable efficiency[ (32.27±7.56) % ] were specifically able to kill A20 tumor cells (The apoptosis rate was 24.3% at 48 h) . In vivo detection showed a non-significant decrease in the percentage[ (1.83±0.58) % ] of splenic CD19(+) cells 6 days after mCD19 CAR-T cell infusion. A marked clearance in bone marrow and spleen appeared on day 12 compared with the A20 group, and this difference was statistically significant[spleen: (0.36±0.04) % (47.00±13.46) % , <0.001; bone marrow: (1.82±0.29) % (37.30±1.44) % , <0.0001]. Moreover, mCD19 CAR-T cells were distributed in high proportions in the peripheral blood, spleen, and bone marrow[ (2.90±1.12) % , (4.96±0.80) % , (13.55±1.56) % ]. This study demonstrated an optimized activation system and the optimal infection time of CD3(+) T cells. Furthermore, stable constructed mCD19 CAR-T cells showed a remarkable killing ability in vitro and in vivo.
为优化小鼠CD3(+) T细胞的体外刺激和激活系统，探索CD3(+) T细胞的最佳感染时间以建立小鼠CD19嵌合抗原受体T细胞（mCD19 CAR-T），并验证其体内外杀伤效果。采用磁珠法分离纯化脾CD3(+)T细胞，将细胞分别培养于可溶性抗CD3/CD28、PMA+离子霉素和包被抗CD3/CD28中。分别于8、24、48和72小时后通过流式细胞术评估细胞活化和凋亡情况。将小鼠CD19抗体的ScFv质粒转染至plat-E细胞以包装逆转录病毒。感染活化的CD3(+) T细胞构建小鼠特异性CD19嵌合抗原受体T细胞（mCD19 CAR-T），并将mCD19 CAR-T细胞与B细胞淋巴瘤细胞系A20进行体外共培养。通过流式细胞术检测A20的特异性毒性，并将mCD19 CAR-T细胞注入淋巴瘤小鼠模型中检测其杀伤效果和分布情况。包被抗CD3/CD28对CD3(+) T细胞的激活效果更佳，刺激后24 - 48小时细胞活力良好。成功建立了效率稳定[（32.27±7.56）%]的mCD19 CAR-T细胞，其能够特异性杀伤A20肿瘤细胞（48小时时凋亡率为24.3%）。体内检测显示，输注mCD19 CAR-T细胞6天后，脾CD19(+)细胞百分比[（1.83±0.58）%]下降不显著。与A20组相比，第12天骨髓和脾脏出现明显清除，差异具有统计学意义[脾脏：（0.36±0.04）% 对（47.00±13.46）%，<0.001；骨髓：（1.82±0.29）% 对（37.30±1.44）%，<0.0001]。此外，mCD19 CAR-T细胞在外周血、脾脏和骨髓中的分布比例较高[（2.90±1.12）%，（4.96±0.80）%，（13.55±1.56）%]。本研究证明了优化的激活系统以及CD3(+) T细胞的最佳感染时间。此外，稳定构建的mCD19 CAR-T细胞在体外和体内均表现出显著的杀伤能力。
