CAR-T cells exposed to IL-7 and IL-15 display superior effector function after serial stimulations. (A) CAR-T cells expanded with IL-2 or IL-7 and IL-15 were first stimulated with irradiated CD19⁺ target tumor cells (Raji). Three days after stimulation, cells were collected and subjected to coculture and intracellular cytokine production assays. (B) CAR-T cells were cocultured with target tumor cells at 1:3 E:T ratio. Percentages of residual tumor cells in the culture were determined by flow cytometry by day 3 of culture (n = 4). (C-D) Intracellular IFN-γ staining and degranulation analysis of CAR-T cells after the 2nd consecutive antigen stimulation (n = 4). Minimal (<2%) expression of IFN-γ or CD107 was observed when cells were not stimulated by Raji. (E-F) Intracellular IFN-γ staining and degranulation analysis of CAR-T cells after the 2nd consecutive antigen stimulation after depletion of the DP subset (n = 3).