E g7 ova cell line

All animal protocols were approved by the regional animal study committee of Berlin (Germany, LaGeSo) and all methods were performed in accordance with the relevant guidelines and regulations as published by the study committee.
Six- to eight-week-old female BALB/c mice were obtained from Harlan Winkelmann (Borchen, Germany). The MSC-2 cell line was kindly provided by Professor Ghiringhelli (Institut National de la Santé et de la Recherche Médicale (INSERM) U866, Dijon, France). Cells were passaged at 37 °C in a 5% CO₂ atmosphere in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen GmbH, Darmstadt, Germany) and penicillin/streptomycin (100 U/ml/ 100 µg/ml, Sigma-Aldrich Chemie GmbH, Munich, Germany). The E.G7 OVA cell line was bought from ATCC (ATCC ^®CRL-2113^TM). The RAW264.7 cell line was passaged in the same condition as MSC-2 cell line in the presence of 1 mM sodium pyruvate. T cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen GmbH), penicillin/streptomycin (100 U/ml/ 100 µg/ml, Sigma-Aldrich Chemie GmbH) and 50 µM β-mercaptoethanol (Sigma-Aldrich Chemie GmbH).

Mouse embryonic fibroblasts (MEFs) were derived from 13.5 d.p.c C57BL/6 mouse embryos. MEFs were maintained in DMEM/high glucose (Hyclone), 10% FBS (Natocor) supplemented with 1% nonessential amino acids (NEAA, Gibco). C57BL/6 mouse embryonic stem cells (Biocytogen) were maintained on feeder layers in ES medium containing DMEM/high glucose, 15% FBS (Gibco), 1% NEAA, 1% GlutaMAX (Gibco), 1% Sodium Pyruvate (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1 μM PD0325901 (Selleck), 3 μM Chir99021 (Selleck) and 1000 U/mL LIF. The OP9-DL1 cells (GFP⁺) were maintained in α-MEM (Gibco) supplemented with 20% FBS (CellMax). The AFT024 cell lines (ATCC) were maintained in DMEM/high glucose, 10% FBS (Natocor) supplemented with 0.1 mM β-mercaptoethanol and 1% Sodium Pyruvate. HEK293T (ATCC) and Plat-E (Cell Biolabs, Inc) cells were maintained in DMEM/high glucose supplemented with 10% FBS (Natocor). E.G7-OVA cell line (ATCC) was cultured in RPMI 1640 (Gibco) supplemented with 10% FBS (Natocor), 1% GlutaMAX, 1% Sodium Pyruvate, and 0.1 mM β-mercaptoethanol.

1 × 10⁶ mPD-1-deleted E. G7-OVA Cell Line (ATCC, CRL-2113, RRID:CVCL_3505) reconstituted by hPD-L1 were subcutaneously inoculated in 100 μl PBS in the dorsal region of Rag2^−/− mice. Tumors were allowed to grow for 8 to 10 days before treatments (tumor area between 90 and 400 mm²). Tumor size was blindly measured using calipers. Mice received 100 μl PBS containing 1.5 × 10⁶ activated Pdcd1^−/− OT-I TCR-Tg CD8⁺ T cells expressing hPD-1 by intravenous injection in the tail vein. Two days later, mice were injected intraperitoneally with nivolumab (MCE, HY-P9903, RRID:AB_2810223) or durvalumab (MCE, HY-P9919) at 0.001 mg/kg or 0.1 mg/kg four times every 2–3 days.