CAR-T cells exposed to IL-7 and IL-15 display superior proliferative capacity after serial stimulations. (A) CAR-T cells expanded with IL-2 or IL-7 and IL-15 were stimulated 3 times with irradiated CD19⁺ target tumor cells (Raji) in the absence of exogenous cytokines. After each stimulation, cells were counted and phenotypic analysis was performed by flow cytometry. (B) Cell count of CAR-T cells after each antigen stimulation (n = 4). (C) Ratio of CD4⁺ and CD8⁺ T cells before and after 2 consecutive antigen stimulations (n = 5). (D) Apoptosis of CD4⁺ and CD8⁺ CAR-T cells after antigen stimulation assessed by Annexin-V and 7-AAD staining (n = 3). (E) CAR-T cells were labeled with CFSE before being stimulated by tumor cells. CFSE dilution was determined by flow cytometry on CD4⁺ and CD8⁺ T cells by day 3 of culture. Data shown are representative of 3 independent experiments. (F-G) CD45RA and CCR7 DP T cells were sorted by flow cytometry from IL-7 and IL-15 expanded CAR-T cells (IL-7+15 DP). Negative fraction was also collected as IL-7+15 DP⁽ⁿ⁾. After sorting, IL-2 expanded (IL-2), IL-7 and IL-15 expanded (IL-7+15), IL-7/15 DP, IL-7, and IL-15 DP⁽ⁿ⁾ CAR-T cells were stimulated as in (A). Cell counts (F) and apoptosis on CD4⁺ or CD8⁺ T cells (G) were measured by flow cytometry (n = 3).