Characterization of Tumor-Infiltrating Leukocytes

For characterization of TIL, mice were euthanized at the days specified in the results section. Tongue and flank tumors were collected and digested as previously described [10 (link)]. Purified leukocytes were stained for multi-parametric flow cytometry analysis with a 16-color antibody panel. Cells were blocked with mouse Fc-block, stained with surface markers, fixed and permeabilized with the FoxP3 Fix/Perm Kit (eBioscience, Waltham, MA) followed by staining for intracellular markers. Samples were run in an LSR-II X-20 Fortessa (BD Biosciences, San Jose, CA) at the South Campus Flow Cytometry Core, MD Anderson Cancer Center (Houston, TX) and analyzed using FlowJo version 10 (Flowjo LLC, Ashland, OR). The live/dead fixable aqua dye (Thermo Scientific, Waltham, MA) was used to gate out dead cells and to include only live cells for analysis. Live leukocyte gate was set based on forward and side scatter to include both lymphocytes and larger myeloid cells. Tregs were identified based on CD4⁺Foxp3⁺ expression within the CD3⁺ gate. From CD3⁻ gate, CD11b⁺Gr-1⁺ cells were identified as total MDSC population. The ratio of CD8⁺ T cells to Tregs or MDSC were calculated by dividing the percentage of CD8⁺ T cells with that of CD4⁺Foxp3⁺ or CD11b⁺Gr-1⁺ cells.