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Archive > Volume 32 Issue 4 > 2025,32(4):356-363. DOI:10.3872/j.issn.1007-385X.2025.04.002 Prev Next
Basic Research
Construction and functional study of CD19 CAR-T cells that secrete IL-2 locally
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Abstract:
[Abstract] Objective: To investigate the impact of locally IL-2-secreting CAR-T cells (Syn.CD19.IL-2 CAR-T cells) constructed using a novel single-vector SynNotch system on the function of conventional CD19 CAR-T cells. Methods: Building upon our previously established single-vector SynNotch system, the CD19-specific FMC63 antibody was integrated with an IL-2 expression module to prepare Syn.CD19.IL-2 CAR-T cells through T cell transduction using a self-inactivating retroviral vector. For validation experiments, cells were divided into the Syn.CD19.IL-2 CAR-T cell group and the untransduced T cells group. ELISA was used to detect IL-2 secretion levels after antigen stimulation. The CFSE staining assay was implemented to evaluate the effects of IL-2 secretion on the proliferation of CD19 CAR-T cells when CD19 CAR-T cells were co-cultured with Raji-Luc or SW620-CD19-Luc cells in the presence of Syn. CD19. IL-2 CAR-T cells. Flow cytometry was employed to detect CD69 expression and monitor the activation status of CD19 CAR-T cells when co-cultured with Raji-Luc cells in the condition of Syn.CD19.IL-2 CAR-T cells secreting IL-2. Results: The self-inactivating retroviral vector Syn.CD19.IL-2 CAR were successfully constructed and Syn.CD19.IL-2 CAR-T cells with a viral titer >1 × 10? copies/mL and transduction efficiency of 37.1% were generated. After antigen stimulation, SynNotch CAR-T cells demonstrated significantly elevated IL-2 secretion compared with the untransduced T cell group (P < 0.001). When Syn. CD19.IL-2 CAR-T cells secreted IL-2, the CD19 CAR-T cells exhibited enhanced proliferative capacity and elevated activation level (all P < 0.001). Conclusion: Successfully constructed Syn.CD19.IL-2 CAR-T cells can significantly enhance the proliferative capacity and activation ability of conventional CD19 CAR-T cells.
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