Fig. 3. CD19/20/22CAR T cells have increased immunoactivity and serial killing activity at the single-cell level.

a Schematic illustrating the parameters assessed in the chimeric antigen receptor immune synapse (CARIS). b UPN03 cells were cocultured with NT, CD19CAR, or CD19/20/22CAR T-cells at a ratio of 1:1 for 1 h. After incubation, cells were analyzed for expression of CD3 (BV450), phalloidin (FITC), and 7-AAD using ImageStream. (n = 3) Duplex cells were separated from single cells by DNA contents and aspect ratio of 7-AAD. Intact T and B (BL-ALL) cells were identified by CD3 intensity and area of CD3(−), respectively. Duplex cells containing both T and B cells were selected for further analysis. Formation of immune synapses between T and B cells were defined by length and area of two DNA clusters between duplex cells. The final image exemplifies the characterization of a CARIS. The DNA is shown as red and F-actin (phalloidin staining) as green. The area boxed in white is the immune synapse (IS) area that is quantified in the analysis. Quantification of (c) the area encompassed by the IS (CARIS^area), (d) the intensity of F-actin (phalloidin) at the CARIS (CARIS^density), and (e) the ratio of intensity of phalloidin between T-cells to B-cells (BL-ALL) (CARIS^tension). The frequency of each parameter for NT (gray), CD19CAR (black), and CD19/20/22CAR (red) T-cells is shown in a histogram. 1 × 10⁵ events were assessed; two-way ANOVA was performed for statistical analysis, and p < 0.05 was considered significant. (f–k) CD19CAR or CD19/20/22CAR T-cells were incubated with UPN02 cells at an E:T ratio of 1:3 to assess their cytolysis activity at the single-cell level using a TIMING nanowell assay. (f) Schematic depicting measurements quantified by TIMING assay. (g) Microscopy images representing T^seek, T^contact, and T^death parameters. Scale bars represent 10 μm. (h–j): Quantification of the time spent (h) searching for target by T-cells (T^seek), (i) the duration of contact maintained between an individual CAR T-cell and their first, second, and third target (T^contact), and (j) the time to apoptosis of individual target cells since the start of conjugation (T^death) at an E:T ratio of 1:3. Each data point represents a single effector cell. ****p < 0.0001, **p < 0.01, *p < 0.05, ns = > 0.05; Kruskall–Wallis test with Dunn’s multiple correction. Data are presented as mean ± 95% confidence interval. (k) Pie chart comparing proportion of CD19CAR and CD19/20/22CAR T-cells able to kill multiple targets when plated at an E:T ratio of 1:3. > 165 individual wells were analyzed for each effector cell type.