Fig 2: CD39+ personalized tumor-reactive CD8+ T cells generated from organoid-T cell co-culture system exerted stronger antitumor activity(A) Experimental design. HCC samples were obtained from a needle biopsy of primary tumors. Organoids were established and co-cultured with autologous peripheral blood lymphocytes (PBLs). After 1 week of co-culture, CD39+/- tumor-reactive CD8+ T cells were expanded and sorted by FACS separately. CD39+/- CD8+ T cells were then co-cultured with autologous tumor organoids for 24 h. The antitumor effect was observed by measuring the apoptotic cells within the tumor organoids. (B) Frequency of CD39 gated on tumor-reactive CD8+ T cells after 1 week of co-culture was evaluated by flow cytometry. No organoids added was used as NC. (C) Tumor-reactive CD8+ 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 image per hour for 24 h. Representative images for 1 representative patient were shown. Scale bar, 300 µm. (D and E) Summaries of quantitative statistics of (C). Apoptotic cells (green) and infiltrating tumor-reactive CD8+ 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 T cells at 0 h was defined as 0. ***p < 0.0005 (1-way ANOVA). All data are means ± SEMs. Error bars represent SEMs of 3 view fields from each patient. (F) Representative flow cytometry analysis of organoids apoptosis by caspase3/7 probe (FITC). (G–I) Representative flow cytometry analysis and relative quantification of IFN-? production and CD107a expression of CD39+/- CD8+ T cells. Error bars represent SEMs of 3 biological replicates.

Fig 3: HBVs protein was abundant on HCC organoids, and HBVs-CAR-T cells were constructed for cellular immunotherapy(A) Bright-field microscopy images, H&E staining, and immunofluorescence staining of HCC markers AFP (red) and HBVs (green) for organoids were shown separately. Nuclei were stained with DAPI (blue). Scale bars, 200 µm (bright field), 100 µm (H&E and immunofluorescence). (B) Flow cytometry was used to analyze the frequency of HBVs protein expression on HCC organoids of 3 patients. (C) Schematic representation of the lentiviral vectors carrying a HBVs-specific 3rd-generation CAR moiety. (D) Western blotting and immunofluorescence staining for HBVs in HepG-2.2.15 cells; HepG-2 cells served as the mock cells. (E) Immune response was evaluated by measuring the number of IFN-? production and spot-forming cells (SFCs) per 104 HBVs-CAR-T cells (E:T = 4:1). CAR-T cells added with PHA served as PC (positive control) and CAR-T cells alone as NC (negative control). (F) Cytotoxic activity of HBVs-CAR-T cells on HepG-2.2.15 targets cells were detected by LDH assay (n = 3, 3 healthy donors). (G) After co-culture with HepG-2.2.15 cells for 24 h (E:T = 4:1), CD107a+ and CD107a- HBVs-CAR-T cells were obtained by flow sorting, and their transactional profiling was analyzed by RNA sequencing (RNA-seq). Heatmap of RNA-seq showed the expression of genes involving in cytotoxicity, adhesion, and chemotaxis in different effector sub-populations, with p < 0.01. RNA expression levels were indicated with a red/blue scale for high and low expression levels, respectively.

Fig 4: 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 5: The CD39+ HBVs-CAR-T cells exerted an enhanced effector function and a downregulation of inhibitory receptors(A) Representative flow cytometry plots of CD107a and IFN-? gated on CD39+/- HBVs-CAR-T cells after being co-cultured with autologous tumor organoids. GP120-CAR-T cells served as NC. (B and C) Relative quantification of IFN-? and CD107a expression of CD39+/- CAR-T cells. Error bars represent SEMs of 3 biological replicates. (D) Flow cytometry analysis of inhibitory receptor expression of CD39+/- HBVs-CAR T cells.