Fig. 3:. CAR T cell cooperativity augments insufficient clonal tumour-lytic potential.
a, Single-cell time-lapse imaging cytotoxicity assay. Time from the formation of CAR T cell–target cell conjugate (start point, T0) to target cell death (determined by positivity of the propidium iodide (PI) dye; TPI) recorded over 24 h in wells containing 1 CAR T cell and 1 tumour cell (1:1 effector:target (E:T) ratio). CTV, CellTrace Violet. b, Left, percentage of wells with target killing. Right, time from conjugate formation to propidium iodide influx in target cells (lytic conjugates). c, Left, percentage of non-lytic conjugate formation. Right, duration of non-lytic conjugates. d, Cytotoxic time-lapse imaging with two CAR T cells and one tumour cell (2:1 E:T). Time from first conjugate formation (start point, T0–1) to target cell death (TPI) is indicated as ΔT1. Time from second conjugate formation (start point, T0–2) to target cell death (TPI) is indicated as ΔT2. e, Percentage of target killing in wells at a 2:1 E:T ratio. f, Ratio of observed killing (Pobs.2/1) to estimated killing (Pest.2/1) in wells at a 2:1 E:T ratio. g, Percentage of target killing due to engagement of two CAR T cells with one tumour cell. h, Time from the formation of first conjugate (ΔT1) and second conjugates (ΔT2) to propidium iodide influx in target cells. In b, c, e–h, n = 5 donors; in b, c and h, time is indicated in minutes. i, Percentage of target killing in wells at 1:1 (left) and 2:1 (right) E:T ratios. WT denotes wild-type NALM6 cells. Med and Low denote NALM6med and NALM6low cells, respectively. n = 3 donors. In b, c, n ≥ 197, e–h
n ≥ 242, i, n ≥ 70 (ratio 1:1) and n ≥ 42 (ratio 2:1) individual wells were examined. P values were determined by two-sided two-sample test of proportions (b (left), c (left), e, g and i), two-sided unpaired t-test (b (right), c (right) and h) or two-sided Mann–Whitney U-test (f). Data are mean ± s.e.m.