Isolation of Tumor-Infiltrating Lymphocytes and Flow Cytometry Analysis

Flow cytometry experiments were performed on a separate cohort of animals that was not included in the survival study. Mice were sacrificed at post-implantation day 14 and intracranial tumors were harvested. Tumor-infiltrating leukocytes (TIL) were isolated by generating a single cell suspension through mechanical dissociation of the tumor tissue. Myelin was removed using a 30% Percoll density gradient. Red blood cells were removed using ACK lysis buffer. The resulting cell pellet was stained with fluorophore-conjugated antibodies to CD45, CD3, CD4, CD8, NK1.1, CD11b, Gr-1, and Zombie NIR (live/dead). All antibodies were obtained through BioLegend (San Diego, CA). Flow cytometry was performed on a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA). Analysis was performed through FlowJo software (BD Biosciences). Statistical analysis was performed using the Student t-test in Prism (GraphPad Software, San Diego, CA).

Live CD45⁺ TIL were subgated into lymphoid and myeloid subsets to determine relative frequency among total TIL. Lymphoid cell populations were defined as CD4 or CD8 T cells (CD3⁺ NK1.1^- CD11b), NK cells (CD3^- NK1.1⁺ CD11b^-), or NKT cells (CD3⁺ NK1.1⁺ CD11b^-). Myeloid cell populations were defined as CD3^- NK1.1^- CD11b⁺, and further gated on granulocytic MDSC (Gr-MDSC; CD11b^lo Gr-1^hi), monocytic MDSC (M-MDSC; CD11b^hi Gr-1^lo), or tumor-associated macrophage/microglia (CD11b⁺ Gr-1^-). Gating on resting microglia (CD45^lo), activated microglia (CD45^int), and tumor-associated macrophage (CD45^hi) was performed on the CD11b⁺ Gr-1^- subset.