To demonstrate in vitro anti-tumor reactivity, IFNγ secretion was measured following co-incubation of CD19 CAR T cells with target cells (18 (link), 21 (link)–23 (link)). Un-transduced T cells served as negative control. The following CD19-expressing target cell lines were used: NALM-6 (acute lymphoid leukemia); Toledo (B cell diffuse large cell lymphoma) and CD19-K562 (K562 cell line transduced to express CD19). The CD19-negative cell line CCRF-CEM (T cell leukemia) served as negative control. All tumor lines were kindly provided by Dr. Steve Rosenberg, NCI.
The co-culture was performed with an effector to target ratio of 1:1 (1×105 each) in a total of 200μl medium overnight at 37°C. Supernatant was collected, if necessary diluted and IFNγ secretion was determined by ELISA (Human IFN- ELISA MAX Deluxe Set, Cat. 430106, BioLegend, San Diego, CA). Measurements were performed in triplicates.
The co-culture was performed with an effector to target ratio of 1:1 (1×105 each) in a total of 200μl medium overnight at 37°C. Supernatant was collected, if necessary diluted and IFNγ secretion was determined by ELISA (Human IFN- ELISA MAX Deluxe Set, Cat. 430106, BioLegend, San Diego, CA). Measurements were performed in triplicates.