Video time-lapse microscopy to evaluate tumor-cell killing by UCB-derived T cells. (A) Relative binding of human CD19-specific mAb by flow cytometry expression of truncated CD19 on parental U251T (red) and transfected U251T (green), compared with isotype-matched mAb binding to transfected U251T (dashed black line). The CD19 expression of transfected cells was 87% as measured by Overton analysis (FCS Express Version 2; De Novo Software, Ontario, Canada). These tumor cells were used in the VTLM studies described for panel B. (B) Net change in number of adherent tumor cells over time (240 minutes, 960 frames) cocultured with UCB-derived T cells. Tumor-cell divisions were observed in all experimental conditions (CD19R^± T cells cocultured with CD19^± tumor cells). Killing of tumor cells, represented by a net decrement in the number of tumor cells (below the x-axis), was only observed in flasks of CD19R⁺ T cells cocultured with CD19⁺ tumor cells. The data were compiled from 3 video time-lapse sequences acquired with the combination of CD8⁺ CD19R⁺ T-cell clone and CD19⁺ tumor cells, and 5 video time-lapse sequences were acquired of negative controls (parental CD19^– tumor cells and CD8⁺ UCB derived T-cell clone that did (CD19R⁺) and did not (CD19R^–) express the chimeric immunoreceptor). (C) Histogram of residence time (minutes) of CD8⁺ CD19-specific T-cell clone on CD19⁺ tumor cell before a CD19⁺ U251T tumor-killing event was recorded. Two-minute bins were used, and 145 T-cell contact events were evaluated. (D) VTLM of UCB-derived CD19R⁺ T cells cocultured with CD19⁺ tumor cells. Two images are shown (at 0 and 55 minutes). Red “k” indicates killing event, green “d” indicates cell division. Online material includes a movie (Video S1) of the VTLM (at 1 × and 2 × magnification) showing the coculture of CD19-specific T cells with CD19⁺ tumor cells over 120 minutes (part I) and first 60 minutes (part II).