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. 2019 Oct 25;10:2514. doi: 10.3389/fimmu.2019.02514

Figure 1.

Figure 1

ScDb-redirected T-cell killing of melanoma cell lines. (A) Growth inhibition assays performed using nine different batches of PBLs (each derived from a different healthy donor) in the presence or absence of scDb. TRAIL-R2-expressing Me64, Me41, and Me15 and TRAIL-R2-negative MDA-MB-468 were used as target cells treated with 0.5 μg/mL of scDb and non-activated PBLs at an E:T ratio of 5:1 for 48 h (left panel) or 96 h (right panel). Each point represents a single experiment. Bars represent the mean of all nine experiments; error bars, SD. (B) Percentage of apoptosis (early + late apoptosis, annexin V+) of two sTRAIL-sensitive melanoma cell lines (top, Me15 and Me41) and two sTRAIL-resistant melanoma cell lines (bottom, Me71 and Me79) co-cultured for 72 h with 0.1 μg/mL scDb, non-activated PBLs, or both at E:T = 5:1. (n = 3; error bars, SD). (C) Representative plot of data shown in (B). (D) Tumor cells were treated for 4 and 16 h with pre-activated PBLs in the presence or absence of TRAIL-R2xCD3 scDb. Irrelevant scDb Mec14xCD3 was used as control. The graphs show the percentage of direct cell lysis as the mean ± SD of three experiments. (E) Immunofluorescence was performed using Me15 cells loaded with calcein-AM (green) and grown for 4 h with pre-activated PBLs stained using a PKH26 Red Fluorescent Cell Linker Kit in the absence (left panels) or presence (right panels) of 0.5 μg/mL TRAIL-R2xCD3 scDb. 0.5 μg/mL irrelevant scDb Mec14xUCHT1 was used as control (central panels). The pictures were taken at the end of the incubation time. Statistical analysis in (A,B,D) by one-way ANOVA followed by Tukey's post-test. *p < 0.05, **p < 0.01, ***p < 0.001; ns: not significant.