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Published on: August 18, 2023
[Construction of specific artificial antigen-presenting cells for in vitro activation of CD19 chimeric antigen
Yao-Jun Peng1, Qi-Yan Wu, Hong-Yu Liu
1Cancer Center Key Laboratory, General Hospital of PLA, Beijing 100853, China.E-mail: pengyaojun@yeah.net.
Objective:
To construct CD19-specific artificial antigen-presenting cells (aAPCs) for in vitro activation and expansion of CD19 chimeric antigen receptor (CAR)-modified T cells (CD19-CAR-T) and investigate their cytotoxic effect.
Methods:
CD19-specific aAPCs (NIH3T3-CD19/86, NIH3T3-CD19/86/137L) expressing costimulatory molecules CD86 and/or CD137L were prepared on the basis of NIH3T3 backbone cells by lentivirus-mediated gene transfer. Irradiated CD19-specific aAPCs were co-cultured with CD19-CAR-T cells to activate and amplify CD19-CAR-T cells. The growth curve of CD19-CAR-T cells was determined by trypan blue exclusion assay, and CD19CAR expression and phenotype on CD19-CAR-T cells were detected by flow cytometry. The in vitro cytotoxicity of CD19-CAR-T cells against the target cells was evaluated by bioluminescence-based cytotoxicity assay.
Results:
Flow cytometry showed that NIH3T3-CD19/86 and NIH3T3-CD19/86/137L expressed high levels of CD19, CD86 and/or CD137L. Both NIH3T3-CD19/86 and NIH3T3-CD19/86/137L cells could amplify CD19-CAR-T cells efficiently, but NIH3T3-CD19/86/137L cells had better amplification effect. After 14 days of co-culture with NIH3T3-CD19/86/137L cells, the number of CD19-CAR-T cells was significantly greater than that of NIH3T3-CD19/86 cells (P<0.05), and the proportion of CD19-CAR-T cells in the total T cells increased significantly (P<0.05). CD19-CAR-T cells amplified by CD19-specific aAPCs produced target-specific cytotoxicity and were able to specifically kill CD19-positive target cells. About 20% central memory T cells were present in the final products expanded by NIH3T3-CD19/86/137L.
Conclusion:
We successfully prepared CD19-specific aAPCs that can specifically amplify functional CD19-CART cells in vitro, which facilitates the acquisition of clinical-scale high-quality CD19-CART cells.
