Figure 1.

PD-1 downregulation enhances the antitumor function of CAR T cells

(A) Schematic representation of the lentiviral two-in-one vector carrying a CD19-CAR and shRNA expressing module. (B) PD-1 expression levels of CAR T cells with different shPD-1 candidates as determined by flow cytometry on day 2 after stimulation with gamma-irradiated Nalm-6-GL cells. Gray denotes the isotype control. Data are the pooled mean ± SD from three independent experiments, each performed in triplicate. (C) Cell counts from the homeostatic expansion of LNGFR⁺ CAR T cells with PD-1 downregulation candidates on days 3 and 6 after cell seeding. Data are the pooled mean ± SD from three independent experiments performed in triplicates. (D) The effects of hH1-, hU6-, and mU6-shPD1 on PD-1 expression was analyzed by flow cytometry 2 days after stimulation with gamma-irradiated K562-CD19 cells. Data are the pooled mean ± SD from two independent experiments performed in triplicates. (E) Cell counts from the homeostatic expansion of CAR T cells with each RNA Pol III promoter after LNGFR⁺ isolation on days 3 and 6 after cell seeding. Data are the pooled mean ± SD from two independent experiments performed in triplicates. (F) CAR T cells were incubated with GFP-expressing Nalm-6-GL or Nalm-6-GL-PD-L1 cells at a 1:1, 0.3:1, and 0.1:1 effector-to-target (E:T) ratio. GFP intensity was measured every 2 h using the IncuCyte S3 live cell imaging system. The relative percentage of total integrated GFP intensity was calculated as (GFP intensity at each time point/GFP intensity at 0 h) × 100. Representative mean ± SD from two independent experiments performed in triplicates. (G) CAR T cells were incubated with gamma-irradiated K562-CD19 or K562-CD19-PD-L1 cells at a 1:3 E:T ratio and counted on day 7. Data are the pooled mean ± SD from two independent experiments performed in triplicates. (H) 3 × 10⁵ 19GBBz or 19PBBz T cells were incubated with 9 × 10⁶ gamma-irradiated Nalm-6-GL-PD-L1-CD80 cells every 6 days (first and second stimulation) and supernatants were collected on day 2 after each respective stimulation. Data are the pooled mean ± SD from four independent experiments. (I) 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) and n = 5 mice (19BBz, 19GBBz, and 19PBBz). (J) PD-1 expression levels of CAR T cells from Nalm-6-GL-PD-L1-bearing mice at day 43. Gray denotes isotype control. Data are the mean ± SD from three mice per group. Statistical analysis was done by one-way ANOVA for (C), (E), (F), and (H) and an unpaired two-tailed t test for (G) and (J). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant.