CD19嵌合抗原受体T细胞制备条件优化及其体内外杀伤作用研究

OBJECTIVE: 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. METHODS: 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. RESULTS: 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)％ vs (47.00±13.46)％, P<0.001; bone marrow: (1.82±0.29)％ vs (37.30±1.44)％, P<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)％]. CONCLUSION: 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.