In vitro cytotoxicity assays

MC38 cells were seeded at 34,000 cells per 24 well and pulsed with Ova (1 ng/ml) or scrambled peptide 24 hours after seeding. Pulsed cells were cultured with activated CD8⁺ T cells at the indicated E:T ratios. After 24 hours, nonadherent tumor cells were washed away with PBS and cell viability was assessed. Where indicated, a neutralizing TNFα antibody or isotype control antibody was added at concentrations of 10 or 20 μg/ml.

Human ZR-75-1 cells were seeded at 100,000 cells per 24 well. After 24 hours, cells were incubated with T cells (isolated from human PBMCs) at the indicated E:T ratios for 24 hours in the presence of CD3 bispecific antibodies (12 ng/ml) (control or antitumor antigen) in the absence or presence of 5 μM autophagy inhibitor SAR405.

The effects of TNFα, TRAIL, doxorubicin, or paclitaxel on cell viability were assessed after 24-hour incubations at the indicated concentrations. The effects of 5 μM autophinib or SAR405 on TNFα (10 ng/ml)– or TRAIL (10 or 50 ng/ml)–induced killing were assessed after 24-hour incubations unless otherwise indicated. In all cases, cell viability was measured using cell counting kit-8 (CCK-8) reagent, which is reduced by dehydrogenase activities in cells to give a yellow-colored formazan dye (Dojindo). The absorbance was measured using a SpectraMax M3 microplate reader (Molecular Devices).