Fig 2: Dual CAR-T cells activate more efficiently than single CAR-T cells at low antigen densities. (a) Schematic of CAR-T cell production from healthy donor PBMC samples (n = 3 biological donors) with mCherry-CAT-CD19-BBZ-CAR and eGFP-9A8-CD22-BBZ-CAR lentiviral vectors producing four different cell types: untransduced T cells (blue), aCD19 CAT CAR-T cells (red), aCD22 9A8 CAR-T cells (green), and dual aCD19 CAT and aCD22 9A8 CAR-T cells (purple). (b) FACS results to measure the transduction efficiency of the three healthy donor PBMC samples (HD1, HD2, and HD3) with mCherry-CAT-CD19-BBZ-CAR and eGFP-9A8-CD22-BBZ-CAR lentiviral vectors. (c) Schematic of SLB system functionalized with biotinylated and His-tagged CD19 and CD22, and His-tagged ICAM-1, interacting with a dual 9A8 and CAT CAR-T cell. (d) Confocal images of CAR-T cells stimulated with the SLB at increasing ligand densities of CD19 and CD22 in molecules/µm2. Showing DAPI staining for all cells (blue), GFP staining for 9A8-positive CAR-T cells (green), mCherry staining for CAT-positive CAR-T cells (red), and pERK staining for activated cells (pink). Scale bar is 250 µm. (e) Analysis of confocal pERK data representing % of cells expressing pERK at each ligand density on the SLB for CAT (red), 9A8 (green), and dual (purple) CAR-T cells for HD1, HD2, and HD3 biological donors. CAT and dual CAR T cells were plotted against CD19 ligand density, while single 9A8 CAR T cells were plotted against CD22 density. Each datapoint is from one biological donor with cells taken from 10 fields of view (FOVs), with a separate graph for each biological donor (HD1, HD2, and HD3). (f) Analysis of confocal pERK data representing % of cells expressing pERK interacting with SLB functionalized with ICAM-1 or anti-CD3/anti-CD28 with ICAM-1 on the SLB for untransduced T cells (blue), and dual (purple), CAT (red), and 9A8 (green) CAR-T cells. Each black circle represents one experimental repeat from a different biological donor, with bars representing median with interquartile range shown as error bars. Statistical comparisons were performed using two-tailed nonparametric Mann–Whitney U tests (Wilcoxon rank-sum tests), with Benjamini–Hochberg (BH) correction for multiple comparisons. P values above 0.05 were considered not significant. FACS, fluorescence-activated cell sorting.

Fig 3: Live-cell imaging reveals persistent peripheral actin protrusions in antigen-stimulated CAR-T cells, but normal contraction dynamics via TCR stimulation. (a) Representative still frames from live-cell imaging of untransduced Jurkat T cells (blue, top) and CAR-expressing Jurkat T cells (grey, middle and bottom) labeled with SiR-actin and interacting with SLB. SLBs were functionalized with anti-CD3/anti-CD28 and ICAM-1 to stimulate untransduced T cells (top) or CAR-T cells via the endogenous TCR (bottom), or with CD19/CD22 ligands and ICAM-1 to stimulate CAR-T cells via cognate antigen engagement (middle). Images correspond to the basal synapse plane at specific time points following initial contact. Scale bars: 5 µm. (b) Quantification of normalized cell perimeter over time for untransduced T cells stimulated with anti-CD3/anti-CD28 and ICAM-1 SLBs (blue), CAR-T cells stimulated via cognate antigen engagement on CD19/CD22- and ICAM-1–functionalized SLBs (grey), and CAR-T cells stimulated via TCR activation on anti-CD3/anti-CD28 and ICAM-1 SLBs (dark grey). The perimeter-to-area ratio reports the temporal dynamics of peripheral actin protrusions during IS formation. Corresponding full time-lapse sequences are shown in Videos 1, 2, and 3. All box plots show median (center line), interquartile range (IQR; box), and full data range (whiskers). For untransduced T cells, n = 8 cells; for CAR T cells, n = 8 (CD19/CD22- and ICAM-1–functionalized SLBs) and 9 cells (anti-CD3/anti-CD28 and ICAM-1 SLBs). n represents individual cells (technical replicates). Statistical comparisons were performed using two-way ANOVA followed by Dunnett’s multiple comparisons test. P values below 0.05 (relative to 1-min interval) were considered significant using the following notation *P < 0.05, **P < 0.01.