(A) Schematic overview of the dynamic profiling and image analysis workflow of CAR T cell multifunctionality using TIMING. We evaluated the interaction between CAR T cells and NALM-6 cells as tumor cells on arrays of nanowells using TIMING. (B) A representative example of a multifunctional 19-28z T cell that participated in killing and secreted IFN-γ. TIMING is utilized to quantify T cell–intrinsic behavior such as directional migration and the kinetics of the interaction, leading to induction of apoptosis within tumor cells. After the TIMING assay, the IFN-γ molecules captured onto the beads during TIMING are revealed by using appropriate fluorescently labeled antibodies. Time is displayed as hours and minutes. Scale bar: 20 μm. (C) Schematic description of kinetic parameters measured in TIMING experiments. t_Seek, time taken for the effector cell to conjugate to the tumor cell. (D and G) Cumulative contact duration between effector and tumor cells leading to different functional outcomes. Effector cells that only secrete IFN-γ (monofunctional) exhibited longer contact duration compared with cytolytic cells with or without IFN-γ secretion. Data are aggregated from profiling all 5 IPs. HD, healthy donor. (E and H) Comparative assessments of t_Contact and t_Death for all killer 19-28z T cells. (F and I) Out-of-contact migration of the different functional subsets of 19-28z T cells. All data in D–F correspond to E:T of 1:1 and are aggregated from profiling all 5 IPs (1589 T cells). All data in G–I correspond to an E:T of 1:2–5 (to evaluate serial killing) (1178 T cells). Each violin plot represents a minimum of 80 single cells. All P values for all multiple comparisons were computed using Kruskal-Wallis nonparametric tests and pairwise comparisons using a Mann-Whitney U test. Black bars represent the median, and the dotted lines denote quartiles.