Figure 1. Primary anti–PD-1 CAR T cells do not express PD-1 and kill PD-1–expressing cells.

RM CD8⁺ T cells transduced with the 6 anti–PD-1 CAR and analyzed by flow cytometry express EGFRt as a surrogate marker for the CAR (A). Gating on EGFRt⁺ and EGFRt^– cells in the culture revealed a loss of PD-1 expression on CAR T and bystander cells in CAR T cells in comparison with nontransduced T cells (B). PD-1⁺ target cells were generated by transduction of K562 cells with a PD-1 GFP fusion protein–expressing lentivirus followed by sorting for different levels of PD-1 cell-surface expression. PD-1 cell–surface expression was quantified with Quantibrite beads and anti–PD-1 antibody EH12 2H7 (C). Anti–PD-1 CAR T cells kill PD-1⁺ target cells. Primary anti–PD-1 CAR CD8⁺ T cells or nontransduced CD8⁺ T cells were cocultured with K562 PD1 GFP with low, medium, and high PD-1 expression or K562-GFP control target cells at a 1:1 ratio. Cytotoxicity was measured by reduction of GFP⁺ cells using the IncuCyte Live-Cell Imaging System. Average cytotoxicity ± SEM of 1 representative experiment is shown (n = 3). Statistics were analyzed using 2-way ANOVA with Tukey’s multiple-comparisons test at each time point. Results for 72 hours are reported. ****P < 0.0001 (D).