Mouse tumor implantation, irradiation, and TIL isolation

C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). For tumor growth experiments, 150,000 B16 or D4M tumor cells were implanted subcutaneously in 100 µl sterile saline and tumor growth was monitored by measuring tumor diameter with calipers every third day for the duration of the experiment. For intracranial tumors 2 × 10⁴ tumor cells were injected as previously described.⁴⁰ Briefly, cells were implanted into the right frontal lobes of wildtype C57BL/6 mice. For bioluminescence imaging, mice were given d-luciferase (Promega) via i.p. injection before imaging using an IVIS imaging system. Animals were immobilized with isoflurane during this procedure. For antibody-treatment, anti-PD-1 antibody or an IgG control were injected at 150 μg/dose i.p. starting five days before irradiation and continuing every fifth day for the duration of the experiment. For irradiation, animals were immobilized via a single injection of ketamine and xylazine and irradiation was delivered to the mouse head or flank tumor using a Pantak X-ray irradiator. Lead shielding was used to limit radiation exposure to other areas of the body. Four 2 Gy doses were administered daily for a total radiation dose of 8 Gy. Tumor-infiltrating lymphocytes were isolated by disruption of tumor tissue first by mincing with crossed scalpels under sterile conditions followed by enzymatic digestion as described above. Live cells were isolated from debris by centrifugation over a Ficoll gradient prior to staining for flow cytometry or cryopreservation for further analysis. All animals were housed in the Biological Resources Unit of the Cleveland Clinic Lerner Research Institute according to Office of Laboratory Animal Welfare guidelines and experiments were conducted under an approved Institutional Animal Care and Use Committee protocol.