Isolation of immune cells from spleen, lymph nodes, and tumor

Tumor-free mice were sacrificed by cervical dislocation and spleen and lymph nodes were extracted and mashed through a 70-μm strainer. Contaminating erythrocytes were lysed using ACK lysis buffer (Gibco).

For tumor-infiltrating lymphocytes (TIL) from brain tumors, mice were anesthetized and perfused with 20 mL PBS. The right hemisphere was extracted and digested with 50 μg/mL Liberase (Sigma) for 30 minutes and subsequently mashed through 100-μm and 70-μm cell strainers to obtain a single-cell suspension. Myelin was removed using a 30% continuous percoll gradient.

ELISpot was performed as previously described (6). Briefly, wetted ELISpot plates (MAIPSWU10, Millipore) were incubated with 100 μL 15 μg/mL IFNγ coating antibody (AN-18, Mabtech) and incubated overnight at 4°C. Cells from spleen and lymph nodes from vaccinated mice were extracted at day 21 after vaccination and resuspended in RPMI-1640 supplemented with 10% FBS, P/S as above, 50 μmol/L beta-mercaptoethanol (Sigma), 2 mmol/L L-glutamine, 25 mmol/L Hepes, 1 mmol/L sodium pyruvate (all Invitrogen), and 0.1 mmol/L nonessential amino acids (NEAA, Lonza; called T-cell medium, TCM). IFNγ coating antibody was removed and ELISpot plates were blocked with TCM. Three hundred thousand to 600,000 cells were plated, and peptides were added at 10 μg/mL. For positive control, 20 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL ionomycin were used. Plates were incubated for 40 hours. Cells were removed and the plate was incubated with 1 μg/mL biotinylated IFNγ detection antibody (R4–6A2, Mabtech) in PBS with 0.5% FBS for 2 hours at room temperature. The detection antibody was removed and wells were incubated with 1 μg/mL streptavadin–alkaline phosphatase (ALP; Mabtech) in PBS with 0.5% FBS for 1 hour. Streptavidin-ALP was removed and plate was incubated with ALP development buffer (Bio-Rad) until distinct spots emerged. Spots were quantified with an ImmunoSpot Analyzer (Cellular Technology Ltd).