Specific tumor cell lysis by CD27 costimulated FR-specific CAR-T cells. (A-B) CAR-transduced T-cell lytic function was tested in bioluminescent killing assay with a 96-well microplate. FR CAR-T cells killed FR⁺ SKOV3 and A1847 cells but showed no killing of the FR⁻ C30 and AE17 cells at the indicated E/T ratio for ∼ 20 hours, where untransduced T cells (UNT) and CD19 CAR-T cells served as negative controls. Results are expressed as a mean and SD of triplicate wells from 1 of at least 3 separate experiments. (C) T cells were cocultured with CFSE-labeled tumor cells at a 2:1 ratio in the absence of exogenous IL-2. After 5 days of coculture, cells were collected, stained with CD3, and analyzed for CFSE dilution by FACS analysis. Both FR-z and 27z CAR-transduced T cells killed SKOV3 cells, as evidenced by the lack of CD3^neg CFSE^pos cells, but not C30 cells. Nontransduced T cells and CD19-27 CAR-transduced T cells did not kill ovarian cells. (D) In all extended coculture experiments, both FR-z and 27z CAR-transduced T cells eliminated the CD3^neg CFSE^pos FR^pos tumor cells (SKOV3, A1847, OVCAR4, and AE17.FR) but not FR^neg tumor cells (C30 and AE17).