T cell activation, migration, and cytotoxicity assays

CD8⁺ T cells were isolated from the spleens or MLNs using the EasySep Mouse CD8⁺ T Cell Isolation Kit (StemCell Technologies). Where indicated, T cells were first expanded by incubation at a 5:1 ratio with BMDCs that had been pulsed with tumor lysates (100 µg/ml with 10⁶ BMDCs) for 30 min and then irradiated (20 Gy). For direct co-culture experiments, tumor cells were first incubated with the indicated treatment, and this was removed before adding CD8⁺ T cells at a 5:1 T cell:tumor cell ratio. Where indicated, cytotoxicity was assessed after 24 h or 48 h using the CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific) at 1.0 µM and gating on CD45-negative cells. To assess T cell activation by STING knockdown or scramble controls, CRC cells were first pulsed with 1 µg/ml of the SIINFEKL peptide for 30 min. Cells were washed twice before addition of OTI CD8⁺ T cells as above. For chemokine blocking experiments, the antibodies (Table S2) were present throughout the co-culture at 2 µg/ml.

For migration assays, conditioned supernatants (DMEM plus 10% FBS) were collected from CRC cells treated as indicated in figure legends. Tissue culture inserts with 5.0-µm pores (Sarstedt) were precoated with 0.8 mg/ml Matrigel for 2 h at 37°C and rinsed twice with warm media. 10⁵ CFSE-labeled CD8⁺ T cells were added to the upper portion of the insert in 100 µl media containing 2% FBS. 500 µl of conditioned supernatants was added to the bottom well. For chemokine blocking experiments, antibodies were added to the media in the lower chamber 30 min before T cell addition and were present during the entire assay. T cells were allowed to migrate for 2 h, and then cells in the upper insert and bottom well were collected and quantified by flow cytometry. Percentage migrated cells were calculated as follows: percentage migrated cells = (number migrated cells) / (number input cells + number migrated cells).