OT-1 T-Cell Activation and Tumor Cell Killing Assay

For OT-1 stimulation experiments, CD8+ OT-1 T-cells were isolated from the spleens of naive OT-1 mice using CD8+ MACS beads (Miltenyi) according to the manufacturer’s instructions. ImLECs were starved over night in Ham’s F12/DMEM + 1% FBS, pulsed with 1 ng/ml SIINFEKL peptide (AnaSpec) for 1 h, and washed three times with PBS. Subsequently, OT-1 and peptide-pulsed imLECs were cocultured for 24 h at a 10:1 ratio in the presence of 10 µg/ml PDL1-blocking antibody (clone 10F.9G2, Biolegend) or control rat IgG (Sigma). For analysis of T-cell activation, OT-1 cells were harvested and stained with rat anti-CD8-FITC (1:200, clone 53.6-7, BD), rat anti-PDL1-PE (1:200, clone MIH5, eBioscience), and rat anti-CD25-PerCP (1:200, clone PC61, Biolegend). Intracellular staining for IFN-g (1:200, clone XMG1.2 conjugated to APC, Biolegend) was done using the Cytofix/Cytoperm kit (BD) and fixable Zombi-NIR (BioLegend) was used for life/dead discrimination. For imLEC pre-blocking experiments, the PDL1-blocking antibody was incubated with the imLECs together with the SIINFEKL peptide pulse before washing and coculture with OT-1 cells. For tumor cell killing assays, OT-1 cells were stimulated with peptide-pulsed imLECs as described above and subsequently cocultured with B16F10 cells expressing ovalbumin at a 1:5 target:effector ratio for 8 h. Zombi-NIR was used to determine the ratio of dead and life tumor cells. All activation and killing assays were performed in triplicate. Data were acquired on a FACS CANTO and analyzed using FlowJo.