Fig. 2.

Mechanisms of αCD20 MATCH-induced cytotoxicity in Raji cells. (A) Schematic depiction of MATCH at the synapse of a malignant B-cell and an effector T-cell. Classical T cell-mediated cytotoxicity pathways were verified in response to MATCH treatment, with blinatumomab as positive control. (B) Live-cell confocal microscopy (Zeiss 700) of healthy human T-cells (blue) co-cultured with target Raji B-cells (pink) showing MATCH-induced perforin pore formation (green) in target cells after 1 h incubation. (C) Flow cytometry (FACSCanto^™) quantification of MATCH-induced calcium influx in target cells after 1 h incubation with T-cells. Raji cells were pre-loaded with Fluo-3AM calcium indicator (5 μM). Fluorescence was normalized to untreated co-culture Raji cells (grey). (D) Flow cytometry quantification and live-cell confocal microscopy of MATCH-induced mitochondrial depolarization. Mitochondrial depolarization of individual Fab′-MORF conjugates was compared to MATCH using JC-1 dye (4 μM). Overlay of red (healthy mitochondria) with green (depolarized mitochondria) produces yellow. (E) MATCH-induced caspase-3 activation was quantified using a PhiPhiLux^®-G₁D₂ kit (OncoImmunin). A co-culture of healthy T-cells and Raji B-cells was incubated for 3 h with a MATCH dose. Caspase activation was measured using flow cytometry and normalized to untreated co-culture MFI baseline. (F) Determining contribution of Fasligand and granzymes to overall MATCH-induced apoptosis of target Raji cells. Apoptosis was measured using annexin V/propidium iodide staining on flow cytometry. Inhibited T-cells were normalized to non-inhibited control. For FasL contribution, MATCH was administered in combination with an αFasL antibody (NOK-1; BioLegend). For granzyme contribution, MATCH was performed in combination with a protease inhibitor cocktail (Halt^™). All experiments were performed in triplicate. ***p < 0.001, **p < 0.01, *p < 0.05, n.s. not significant by One-Way ANOVA and Tukey test.