Figure 2.
CAR costimulatory domains differentially affect the in vivo persistence of PD-1-downregulated CAR T cells
(A) PD-1 expression level of CAR T cells with CD28 or 41BB costimulatory domains 2 days after stimulation with gamma-irradiated K562-CD19 cells. Numbers denote the geometric mean fluorescence intensity (gMFI) of PD-1. (B) Unstimulated and (C) stimulated CAR T cells were incubated with Nalm-6-GL-PD-L1 cells at a 1:1, 0.3:1, and 0.1:1 E:T ratio and analyzed using the IncuCyte S3 system. Stimulated CAR T cells were generated by coincubation with Nalm-6-PD-L1-CD80 cells for 6 days prior to cytotoxicity assay. Data are the representative mean ± SD from two independent experiments performed in triplicates. (D) CAR T cells were incubated with gamma-irradiated Nalm-6-GL-PD-L1-CD80 or K562-CD19-PD-L1 cells at a 1:3 E:T ratio and counted on day 6 after each stimulation. Data are the mean ± SD from two independent experiments performed in triplicates. (E) NSG mice were injected intravenously with 1 × 106 Nalm-6-GL-PD-L1 leukemia cells. 5 days later, 1 × 106 CAR T cells were injected intravenously. Tumor burden was monitored based on the bioluminescence intensity from the IVIS imaging system. Data are from n = 3 mice (mock), n = 5 mice (19G28z and 19GBBz), and n = 4 mice (19P28z and 19PBBz). (F) The number of CAR T cells in mouse blood was determined on days 20 and 43 after CAR T cell injection. Data are mean ± SD from three mice per group. Statistical analysis for (A)–(D) and (F) was done by one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant.