Optimization of CD19 chimeric antigen receptor T cell establishment and observation of the killing effect in vitro and in vivo
10.3760/cma.j.issn.0253-2727.2022.06.011
- VernacularTitle: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
- Author:
Chunxiao REN
1
;
Xianxian CHEN
;
Li ZHAO
;
Yu TIAN
;
Kailin XU
;
Kai ZHAO
Author Information
1. 徐州医科大学血液病研究所、江苏省骨髓干细胞重点实验室、徐州医科大学附属医院血液科，徐州 221000
- Keywords:
CD19;
Chimeric antigen receptor T cells;
B-cells lymphoma
- From:
Chinese Journal of Hematology
2022;43(6):506-512
- CountryChina
- Language:Chinese
-
Abstract:
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. Method: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.
