At experiment termination of syngeneic murine models, tumors were cut and digested in a digestion cocktail (collagenase and deoxyribonuclease). Harvested cells in aliquots of up to 1 × 106 cells per 100 μl were dispensed into fluorescence-activated cell sorting tubes and stained for flow cytometry according to the standard protocol (20, 22). CD4, CD8, FOXP3, and Treg cells analyses were performed as the following (9): Tumor cell suspensions were incubated with CD16/32 Ab (BioLegend) at 4°C to block FC receptors. Cells were then stained with CD4-FITC (eBioscience, clone GK1.5), CD8a-allophycocyanin (eBioscience, clone 53-6.7), CD3–peridinin chlorophyll protein (PerCP)–Cy5.5 (BioLegend), FOXP3-allophycocyanin (eBioscience), CD25-phycoerythrin (PE) (BioLegend), CD45-PeCP.Cy5.5 (eBioscience), CD44-PE (eBioscience), CD62L-eFluor 450 (eBioscience), interferon-γ (IFN-γ)–PE (PeproTech), or granzyme B–eFluor 450 (eBioscience). Data acquisition was performed with FACSAria I followed by analyses with FlowJo software (Tree Star). To identify Treg cells, the following gating strategies were used: (i) selection of live single-cell leukocytes [side scatter (SSC)–W/H low, anti-CD45+], (ii) selection of CD4+ T cells (anti-CD4+, anti-CD8), and (iii) selection of CD25+FOXP3+ Treg cells (anti-CD25+, anti-FOXP3+). The investigator was blinded to the group and labeling allocation during the experiment. To identify TH17 cells, the following gating strategies were used: (i) selection of live single-cell leukocytes [side scatter (SSC)–W/H low, anti-CD45+] and (ii) selection of CD194+CD196+ T cells (anti-CD194+, anti-CD196+), CD194–Brilliant Violet 421 (BioLegend), and CD196-PE (eBioscience). To identify DCs, the following gating strategies were used: (i) selection of lymphocyte cells, (ii) selection of CD11c+ lymphocytes and CD11c+ eFluor 450 (eBioscience), and (iii) selection of CD103+ T cells (anti-CD103) and CD103-PE (eBioscience). Each data point was performed in triplicate, and each experiment was repeated twice.

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