Fig 2: The downregulation of inhibitory receptors enhances antitumor activity of CD39+ HBVs-CAR-T cells(A) Schematic representation of the lentiviral vectors carrying a HBVs-specific CAR moiety and a cluster of sh-PD-1, sh-Lag-3, and sh-Tim-3. The second structure of this PTL-shRNA cluster was derived from the miR-106b cluster in human genome. (B) CD39+/- HBVs-CAR-T and CD39+/- PTL-HBVs CAR-T were sorted and co-cultured with autologous tumor organoids for 24 h separately. CD39+ PTL-HBVs-CAR-T cells exerted the strongest infiltrating ability compared to CD39+ HBVs-CAR-T cells and CD39-CAR-T cells. CAR-T cells were labeled with CellTrace Far Red (red) and apoptotic cells were labeled with caspase3/7 probe (green). Real-time Biotech imaging system was used to take 1 picture per hour. Scale bar, 300 µm. (C) Representative flow cytometry analysis of organoids apoptosis by caspase3/7 probe (FITC). (D) Summaries of quantitative statistics of (B) were shown. Apoptotic cells (green) and organoid-infiltrated CAR-T cells (red) were calculated by the Spot function of Imaris software based on the size and intensity threshold. The initial number of infiltrating CAR-T cells at 0 h was defined as the base point. **p < 0.005, ***p < 0.0005 (1-way ANOVA). All data are means ± SEMs. Error bars represent SEMs of 3 view fields from each patient.

Fig 3: CD39+ HBVs-CAR-T cells and CD39+ PTL-HBVs-CAR-T cells exerted potent antitumor activity(A) Representative flow cytometry analysis of CD107a and IFN-? gated on CD39+/- CD8+ PTL-HBVs-CAR-T and HBVs-CAR-T cells after being co-cultured with autologous tumor organoids. (B) Relative quantification of IFN-? production and CD107a expression in CD39+/- PTL-HBVs-CAR-T and HBVs-CAR-T cells. Error bars represent SEMs of 3 biological replicates.

Fig 4: CD39+ HBVs-CAR-T cells exerted potent antitumor activity in the HBV+ HCC organoid-T cell co-culture system(A) The CD39+ and CD39- HBVs-CAR T cells were sorted by FACS and co-cultured with autologous tumor organoids for 24 h separately. HBVs-CAR-T cells (red) were labeled with CellTrace Far Red and apoptotic cells (green) were labeled with caspase3/7 probe. Real-time Biotech imaging system was used to take 1 picture per hour for 24 h. GP120-CAR-T cells served as NC. Scale bars, 300 µm. (B) Summaries of quantitative statistics of HBVs-CAR-T cells (red) and apoptotic cells (green). Apoptotic cells (green) and infiltrating CAR-T cells (red) inside the area of organoids were calculated by the Spot function of Imaris software based on the size and intensity threshold. The initial number of infiltrating CAR-T cells at 0 h was defined as 0. *p < 0.05, **p < 0.005, ***p < 0.0005 (1-way ANOVA). All data are means ± SEMs. Error bars represent SEMs of 3 view fields from each patient. (C) Representative flow cytometry analysis of organoid apoptosis by caspase3/7 probe (FITC).

Fig 5: CD39+ HBVs-CAR-T cells and CD39+ PTL-HBVs-CAR-T cells exerted potent antitumor activity in the PDX model(A and B) HCC organoids at 5 × 106 cells were inoculated s.c. into NSG mice. Fifteen days later, various CAR-T cells were infused via tail injection, and GP120-CAR-T cells (NC-CAR-T cells) served as NC. All of the tumors were measured every 5 days (n = 5). (C–E) At day 40, tumors were resected from the mice, followed by digestion and FACS analysis. The percentage of infiltrating CAR-T cells in tumor (C and D) and the percentages of IFN-?+ cells in tumor-infiltrating CAR-T cells (E) were shown among different groups. *p < 0.05, **p < 0.005, ***p < 0.0005 (1-way ANOVA). All data are means ± SEMs.