Figure 3.

Analysis of polyfunctionality and immune synapses of APRIL CAR T cells. (A) Polyfunctionality by intracellular cytokine staining. Pies depict all different combinations of populations expressing 5, 4, 3, 2, 1 or 0 markers, and arcs around pies indicate the analyte detected. Pies represent mean of n=6 donors. Dependent t-test for paired samples was used to compare populations of CAR T cells expressing at least three markers. (B–E) Fixed cell confocal microscopy for GFP (green, tumor cell), α-tubulin (green, showing the microtubule organizing center (MTOC), centrosome), F-actin (red) and DNA (blue, Hoechst). (B) Left panel: Representative confocal microscopy field of view showing NT T cells and MM.1S-GFP-FFLuc tumor cells, scale bar 30 µm. Middle panel: enlarged inset of left panel, arrows marking T cell - tumor cell pairs, scale bar 30 µm, Inset: enlarged view of one T cell (upper cell)–tumor cell (lower cell) pair, scale bar 10 µm. Scheme on the right explains the immunofluorescence. (C) Left column: representative confocal microscopy images for T cell–tumor cell pair without features of an immunological synapse. Right column: representative images with features of an immunological synapse. First row: Merged images. Second row: F-actin, left: no F-actin accumulation at the cell-cell interface, right: F-actin accumulation at the synapse. Third row: alpha-tubulin and GFP. Arrows are pointing toward the centrosome, the MTOC. Scale bars 10 µm. (D) Quantification of % cell pairs with F-actin accumulation at the cell–cell interface. Each data point corresponds to one field of view, mean±SD, Mann-Whitney-U test. (E) Quantification of % cell pairs with the centrosome close to the cell–cell interface. Each data point corresponds to one field of view, mean±SD, Mann-Whitney-U test. (A, D, E) Definition of significance levels: ns=not significant, *p<0.05, ***p<0.001. APRIL, a proliferation inducing ligand; CAR, chimeric antigen receptor; GFP, green fluorescent protein.