Fig. 4

PE8T cells improved antitumor activity to CD19 + PDL1 + cells with reduced cytokine release and affinity for CD19 protein. A. Schematic of PE8T CAR containing variations in the hinge, extramembrane, and transmembrane domains. B. Cytotoxic percentages of K562, K562-PDL1, NALM-6, and NALM-6-PDL1 cells cocultured with mock-T, 2G, and PE8T CAR-T cells at E:T of 5:1 after 6–8 h in vitro. The results are representative of at least three independent experiments with T cells from different healthy donors (left); the proliferation of different effector cells after CFSE-staining, incubation with different target cells (K562, NALM-6, NALM-6-PDL1) in RPMI (10% FBS) for 48 h. The proliferation rate was analyzed by flow cytometry. (right) Results are mean values ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005. C. IFN-γ, IL-21, TNF-α, IL-10, IL-2, and IL-6 production by mock-T, 2G, and PE8T cells. The concentration of cytokines in the media was measured after 24 h coincubation with N(NALM-6) and NL(NALM-6-PDL1) at E:T of 1:1 (mean ± SD, n = 2). Results are mean values ± SD of triplicate from one of two representative independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005. D. Heat map of selected cytokines enriched in genes significantly upregulated or downregulated in 2G CAR-T vs. PE8T CAR-T cells after stimulation with NALM-6-PDL1 cells at E:T of 1:1 for 48 h. For each pathway, a single sample enrichment score was calculated, and the mean was taken per response group. A color gradient ranging from dark blue to dark red indicates the mean normalized enrichment score (ranging from − 2 to + 2) of pathways enriched in induced (red) or repressed (blue) genes. E. The affinity of CD19 protein to different CAR-T cells: 2G > PE8T. EC50 of CAR-T cells binding to CD19 protein was determined by flow cytometry. The results of D and E are mean values ± SD of triplicate from a single experiment. EC50, 50% maximal effective concentration