Fig. 4

Combinatorial antigen-dependent engineered T cells proliferation. a Scheme of in vitro CAR-T cells proliferation. CD19/HER2 CAR-, dual-targeted CAR-T cells equipped with PD-L1 CCR and untransduced T cells were pre-labeled with Cell Proliferation Dye eFluor 670 according to the manufacturer’s instructions. A549, NCI-H292, and CD19⁺NCI-H292 cells expressing PD-L1 or not were pretreated with mitomycin C to obtain replication-incompetent target cells. These pre-labeled T cells were co-cultured with mitomycin C-treated target cells at an E:T ratio of 2:1 in RPMI 1640 medium supplemented with 10% FBS without cytokines. Finally, the proliferation of T cells was assayed by monitoring the dilution of the cell proliferation dye after incubation for 4 days. b Representative histogram presenting the intensity of cell proliferation dye fluorescence of HER2 CAR-T cells equipped with or without PD-L1 CCR. c-d Representative histogram presenting the intensity of cell proliferation dye fluorescence of CD19 CAR-CD4⁺ (c) and CD8⁺ (d) (n = 3)