Fig. 5

The activation, exhaustion, and clonal expansion characteristics of GD2.BBζ CAR-T cells in vitro. a GD2.BBζ CAR-T cells were labeled with CellTrace™ Far Red to evaluate T cell division. They were co-cultured (ratio of T lymphocytes:tumor cells of 20:1) with four different cell lines that were GD2^negative cell (293 T), GD2^low (GAK), GD2^mediate (HMV-II), or GD2^high cell (WM-266-4 cells). CellTrace™ Far Red expression in T cells was analyzed by FACS after 72 h. Only GD2.BBζ CAR-T cells underwent clonal expansion in response to GD2⁺ melanoma cell lines. Data are representative of repeat experiments. b “Stress test” assay was performed with GD2.BBζ CAR-T cell under repeatedly stimulation onto GD2-cell (293T), GD2+ melanoma cell (HMV-II, GAK, WM-266-4) every 24 h in vitro. The population of the CAR-T cell was evaluated by viability and activation markers (CD25), the dead cells were analyzed by Annexin V and 7-AAD staining