Figure 3.

Construction and characterization of CD19-CAR T and CD19-s47-CAR T cells. (A) Schematic representation of parental CD19-CAR and anti-CD47 secreting CD19-CAR (CD19-s47-CAR) constructs. CD19-CAR is successively connected by a CD8α signal peptide, an anti-CD19 scFv, a CD8α hinge, a CD8α transmembrane, a 4-1BB intracellular costimulatory domains and an intracellular CD3ζ domain. CD19-s47-CAR is composed of CD19-CAR linked with anti-CD47 scFv, and a HA tag was also included for detection of the secreted scFv. (B) Representative flow cytometry plots demonstrating CAR expression in human T cell. A fluorescently labeled CAR-specific antibodies (CARGREEN) was utilized to test the transfection efficiency. Untransduced T cells (UTDT) were used as a control. (C) Western blot was performed to analyze the expression of secreted anti-CD47 scFv in the supernatant from T cells. (D) Secreted antibodies in supernatants were enriched by HA immunoprecipitation kit and quantified by BCA quantification (n = 3). (E) Supernatants of CD19-s47-CAR T cells at different days post viral transfection were collected and quantified as in D and the concentration curve was plotted (n = 3). (F) Detection of the binding ability of secreted scFv in the culture medium (CM) to NHL cells by flow cytometry. NHL cells were incubated with culture medium from different T cells for 30 min at 37°C and then stained by anti-HA flow antibody. Representative flow charts are exhibited. (G) Visualization of anti-CD47 scFv binding to Raji cells by immunofluorescence. Scale bar, 50 μm. These experiments were performed at least three times with similar results.