Fig 1: CD20-TCB mediates efficient tumor cell killing through stable synapse formation.a) Viability assay by AnnexinV (AnnV+) and Propidium Iodide (PI+) staining on WSU DLCL2 and OCI-Ly18 cells, 16 hours after co-culture with CD3/CD28 activated CD8+ T cells in the presence of the indicated CD20-TCB concentrations. n = 4 per point. Mean and +/- s.d. are shown. 2way-Anova, *p = 0.05, **p = 0.005, *** p<0.0005. b) 3D reconstruction of representative confocal imaging of synapse formation between CD8+ T cell (green) and tumor cell (blue), mediated by CD20-TCB treatment (red). c) 3D reconstruction of representative confocal imaging of Granzyme B (red) polarization toward the synapse between CD8+ T cell (green) and tumor cells (blue), mediated by CD20-TCB treatment (white). d-e) Confocal live cell imaging quantification of (d) Percentage of T cell-tumor cell contacts lasting more than 20 minutes; (e) CD8+ T cells speed (µm/min). Cells have been treated with +/- CD20-TCB (200 ng/ml), in the presence of WSU DLCL2 or OCI-Ly18 cells as target. Mean and +/- s.d. are shown, n = 3. 2way-Anova, **p<0.005; ****p<0.0001; n.s.: not significant. f-h) Confocal live cell imaging quantification of (f) Percentage of T cell-WSU DLCL2 contacts lasting more than 20 minutes; (g) contact area between T cells and WSU DLCL2, normalized by the total surface of tumor cells and (h) CD8+ T cells speed (µm/min). Cells have been treated with CD20-TCB at the indicated doses. Mean and +/- s.d. are shown, n = 3. 2way-Anova, *p<0.05; **p<0.005; ****p<0.0001; n.s.: not significant. i) Representative confocal imaging of LFA-1 (white) localization at the synapse between T cell (F-actin is shown in green) and target cell (blue). j) Viability assay by AnnV+ and PI+ staining of WSU DLCL2 cells, 16 hours after co-culture with CD3/CD28 activated CD8+ T cells in the presence of the indicated CD20-TCB concentrations and +/- LFA-1 inhibitor (10 µg/ml) or +/- FAS-L inhibitor (10 µg/ml). n = 4 per point. Mean and +/- s.d. are shown. 2way-Anova, *** p<0.0005, **** p<0.0001.