Fig. 1
CAR grafted with PD1 with or without CD8 hinge exhibited different function to PDL1. A. Schematic representation of four CAR variants with different domains: 2G, PD1-S28, PE8HT, and PEPT CAR, showing variations in the hinge, extramembrane, and transmembrane domains. B. Cytotoxic percentages of targeted cells by mock-T and 2G, PD1-S28, PE8HT, and PEPT CAR-T cells were measured after 12–16 h of coculture in vitro. The E:T ratios (2.5:1 and 5:1) represent the ratios of the absolute number of CAR-T cells to target cells. The following cell types were used: CD19-PDL1- cells (K562), CD19 + PDL1- cells (K562-CD19 and NALM-6), CD19 + PDL1 + cells (K562-CD19-PDL1 and NALM-6-PDL1), and CD19-PDL1 + cells (K562-PDL1 and 786o). The number of mock-T cells was the same as in the 2G group. The results shown are representative of at least three independent experiments using T cells from different healthy donors. C. Proliferation of different effector cells was assessed after stimulation with CD19-PDL1+ (786o) and CD19 + PDL1+ (786o-CD19) cell lines. All effector cells were stained with CFSE. Proliferation was analyzed by flow cytometry by measuring CFSE dilution. The red, green, orange, and blue peaks represent cells before stimulation, cells without any additional stimulation after 48 h, cells incubated with 786o cells (at a 1:1 ratio of 105:105) after 48 h, and cells incubated with 786o-CD19 cells (at a 1:1 ratio of 105:105) after 48 h. CAR: chimeric antigen receptor; CFSE: carboxyfluorescein diacetate succinimidyl ester