Supplemental Table 1, Figures 1 - 20 from Individual Motile CD4+ T Cells Can Participate in Efficient Multikilling through Conjugation to Multiple Tumor Cells

Table ST1: Glossary of terms used in the paper Figure S1. Genetic modification and expansion of CAR+ T cells. Figure S2. In vitro expansion of CAR+ T cells. Figure S3. High-throughput cytotoxicity assay for monitoring T-cell target cell interactions in nanowell grids. Figure S4. CD19 expression on NALM-6 tumor cells or CD19+EL4 target cells as determined by immunofluoresecent staining. Figure S5. Composite micrographs illustrating representative examples of the interactions between single CAR+ T cells (E) and one or more NALM-6 tumor (T) cells. Figure S6. Increased probability of killing by individual CAR+ T cells at higher target cell densities. Figure S7. Donut plots summarizing the outcomes of the interaction between individual CAR8 cells and 1-3 CD19+-NALM-6 tumor cells. Figure S8. Timelapse Imaging Microscopy In Nanowell Grids (TIMING). Figure S9. At an E:T ratio of 1:1, identification of subgroups of killer CAR8 cells based on their motility and contact behavior with tumor cells. Figure S10. At an E:T of 1:1, the total duration of conjugation prior to NALM-6 tumor cell killing is no different for the CAR8 cells in the different subgroups. Figure S11. At an E:T ratio of 1:1, CAR8 cells in the S1 subgroup, demonstrate: (A) drop in motility, and (B) increased circularization upon conjugation to NALM-6 tumor cell. Figure S12. At an E:T ratio of 1:1, CAR8 cells in the different subgroups demonstrate different frequencies and kinetics of AICD subsequent to the interactions with NALM-6 cells. Figure S13. At an E:T ratio of 1:2-5, multi-killer CAR8 cells demonstrate no significant differences in their duration of conjugation prior to killing multiple NALM-6 tumor cells. Figure S14. Despite the increased crowding because of higher tumor cell density, multi-killer CAR8 cells displayed greater motility when conjugated to tumor cell in comparison to single-killer CAR8 cells that encountered only a single tumor cell. Figure S15. At an E:T ratio of 1:1, identification of subgroups of killer CAR4 cells based on their motility and contact behavior with tumor cells. Figure S16. At an E:T of 1:1, the total duration of conjugation prior to NALM-6 tumor cell killing is significantly longer for CAR4 cells in S2 subgroup in comparison to subgroups S1 and S3. Figure S17. At an E:T of 1:1, CAR4 cells in S2 subgroup induce apoptosis in tumor cells with delayed kinetics in comparison to CAR8 cells in the S2 subgroup. Figure S18. At an E:T ratio of 1:2-5, multi-killer CAR4 cells demonstrate increased circularization upon contact with one or more NALM-6 tumor cells. Figure S19. The ability of individual CAR4 cells to simultaneously conjugate to multiple NALM-6 tumor cells increases as the number of tumor cells within the nanowell increases. Figure S20. Comparison of the killing efficiency of individual single killer CAR+ T cells (E:T 1:1) with multi-killer CAR+ T cells (E:T 1:2-5) that killed multiple NALM-6 tumor cells.