CD27 costimulation enhances CAR-T–cell survival and expansion in vitro. (A) CFSE-labeled FR-z and 27z CAR-transduced T cells undergo cellular division when cocultured for 5 days with FR^pos SKOV3 cells but not the FR^neg C30 cells. Untransduced T cells (UNT) and CD19-27z CAR-T cells did not dilute CFSE when cocultured with either FR^pos SKOV3 cells or FR^neg C30 cells. (B) FR CAR-T cell expansion was measured in response to stimulation with FR^pos target cells at a 1:2 ratio in the absence of exogenous IL-2. Viable (trypan blue exclusion) cell counting results showed that FR-27z CAR-T cells underwent increased numerical expansion after 10 days, compared with FR-z CAR-T cells. Counts reflect mean ± SD for triplicate cultures of 2 independent assays. (C) Bcl-X_L expression by FR- or CD19-specific CAR CD8 T cells was examined after 3 days of coculture with SKOV3 or C30 cells. Bcl-X_L expression was preferentially increased in FR-27z CAR T-cell populations, compared with FR-z CAR⁺ T cells after stimulation with FR^pos tumor cells. One representative FACS analysis is shown (n = 3). (D) CD27 costimulation protects against antigen-induced cell death. Apoptotic T cells were detected by simultaneous staining with annexin V (AV)–FITC and 7-AAD-PerCP-cy5.5 at 72 hours after indicated tumor cell stimulation. Apoptosis was quantified in percentages of living (AV⁻/7AAD⁻), early apoptotic (AV⁺/7AAD⁻), late apoptotic (AV⁺/7AAD⁺), and necrotic (AV⁻/7AAD⁺) cells. One representative experiment of 3 is shown.