Chimeric antigen receptor (CAR)-based cell therapies have shown impressive efficacy in the treatment of hematological malignancies. Recently, these therapies are being developed for infectious diseases, yet studies targeting fungal infections remain scarce. To identify optimal targets and optimize cellular products, we developed a method to engineer chimeric antigen receptor-natural killer (CAR-NK) cells and evaluated their response to stimulation by fungi. This paper describes a straightforward and robust method for generating CAR-NK cells tailored to fungal targets using the non-viral Sleeping Beauty transposon system. NK-92 cells are transfected with the hyperactive transposase SB100X vectorized as minicircle DNA (MC) along with a plasmid-encoded CAR transposon. Transfection efficiency is assessed 1 week later using flow cytometric analysis. Prior to functional testing, the cells expressing the transgene are enriched using magnetic-activated cell sorting and cultured for 1 more week. To evaluate antigen-specific activation, the engineered cells are co-cultured with Aspergillus fumigatus germ tubes for at least 6 h. Subsequently, the concentration of the secreted interferon-gamma (IFN-γ) is measured using an enzyme-linked immunosorbent assay.
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