Immune infiltrates were analyzed in tumors and tumor-draining lymph nodes of mice bearing WEHI-164 sarcoma, 48 hours after the last injection of either saline or F8-LIGHT. Mice were euthanized and tumors, right axillary and right inguinal lymph nodes were harvested. Tumors were cut into small fragments and incubated in an orbital shaker at 37°C for 30ʹ in RPMI 1640 Medium (Thermo Fisher, 21875034) containing 1x Antibiotic-Antimycotic (Thermo Fisher, 15240062), 1 mg/mL Collagenase II (Thermo Fisher, 17101015) and 0.1 mg/mL DNAse I (Roche, 10104159001). Tumor cells suspensions were passed through a 70 μm cell strainer (Corning) and treated with Red Blood Cells Lysis buffer (Biolegend, 420301) following supplier’s recommendations. Lymph nodes were harvested and smashed on a 70 μm cell strainer (Corning) using the back of a syringe plunger. The resulting tumor and lymph nodes single-cell suspensions were washed in PBS, before incubation with staining reagents. Where appropriate, cells were stained with Zombie Red dye (diluted 1:500 in PBS) for 15ʹ at room temperature, followed by staining with antibodies and tetramers in FACS buffer (0.5% BSA, 2 mM EDTA in PBS) for 30ʹ at 4°C. Cells, which were not stained with Zombie Red, were stained with 7-AAD (diluted 1:100 in FACS buffer) for 5ʹ at 4°C. Intracellular staining with antibodies against FoxP3 was performed using eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher), following supplier’s protocol. Samples were analyzed with CytoFLEX S (Beckman Coulter) and data were processed using FlowJo software (FlowJo, LLC, version 10). A detailed description of the gating strategies can be found in the Supplemental materials (Supplementary Figure 7–9). Total of living cells in the tumor was calculated by subtracting dead cells and debris from the total number of recorded events.

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