Extended Data Fig. 3. Self-engagement of TROG+ CAR-NK cells resulted in NK cell fratricide.

(a and b) Schematic illustrating the single-cell time-lapse imaging cytotoxicity assay. Time was recorded over 5 hours (T₀ –T_300min) from the start of co-culture, (a) where one single cell, or (b) two cells of non-TROG-antigen expressing fresh CAR-NK cells (control; grey) or CAR-NK^TROG+ cells (green) were incubated in each nanowell. For the duration of the assay, the amount of time taken to detect Annexin V influx in the sorted CAR-NK cell was determined as the time taken to induce cell apoptosis. The following Kaplan-Meier curves show the percent (%) of apoptosis in CAR-NK cells during incubation. (c) Schematic representation of the experimental plan: TROG⁺ CAR19-NK cells were purified, and their phenotypic signature were evaluated before and 5 hrs post-culture by mass cytometry. Data from 10,000 cells from 3 donors per condition were merged to create a single UMAP map with eight distinct color-coded clusters that represented the different subsets of CAR-NK cells. Marker expression for each NK cell subset is shown. (d) UMAP plots showing the expression of TROG-antigen (tCD19; left) on CAR-NK cells before and after 5 hrs of culture with their sibling cells; the contour plots show the prevalence of each CAR-NK cell subset before and after 5 hrs of culture, with the percentage of each subset also indicated (right).

P value was determined by Log-rank test in panels a,b.