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Fig. 4 | Journal of Hematology & Oncology

Fig. 4

From: Accurate control of dual-receptor-engineered T cell activity through a bifunctional anti-angiogenic peptide

Fig. 4

sdCAR-engineered CD8+ T cells yield antigen-specific and titratable killing of cognate target cells in vitro. a sdCAR-engineered CD8+ T cells eradicated cognate target cells expressing MSLN antigen and integrin αvβ3 with an “AND logic gate” strategy. b Specific cytotoxicity of sdCAR-engineered T cells. sdCAR-T cells combined with FHBM have significant cytotoxicity for MSLN+ K562 cells. c Representative flow cytometry data for cytotoxicity of engineered-CD8+ T cells. T cells were incubated with a mixture of cognate target cells (GFP+) and non-cognate target cells (mCherry+). After a 22-h incubation, we quantified the abundance of both surviving cognate and non-cognate tumor cells. The normalized percentage of surviving cognate target cells is expressed as the percentage of MSLN+ K562 cells (Q1) divided by that of CEA+ K562 cells (Q4). d Cytotoxicity mediated by sdCAR-CD8+ T cells in a 22-h experiment. The lower surviving percentage of MSLN+ K562 cells indicated a significant degree of cognate target cell killing by sdCAR T cells only upon the addition of FHBM. (n = 3, error bars denote standard deviation.) e Representative fluorescence images of target cells. After 22 h of interaction, we observed mixtures of cognate and non-cognate target cells incubated with engineered-CD8+ T cells by fluorescence microscopy. f Time course of cognate target cell killing by sdCAR-T cells. The sdCAR-T cell cytotoxicity was not monitored in the absence of switch molecules for the first 4 h (−). However, sdCAR-CD8+ T cells exerted high cytotoxicity for cognate target cells only in the presence of FHBM (+). (n = 3, error bars denote standard deviation.) g Effect of the switch molecule dose on the cytolytic capacities of sdCAR-T cells. The level of cognate target cell killing was correlated with concentrations of FHBM. When the concentration of the switch molecule was increased to more than 100 pM, there was no further increase of T cell cytotoxicity. (n = 3, error bars denote standard deviation)

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