Most subjects included in the study were Caucasian. The study was approved by the local ethics committee, and all patients in the study gave informed consent before tissue donation. Peripheral blood mononuclear cells (PBMCs) were isolated Inhibitor Library via Ficoll density gradient centrifugation. Single cell suspensions from TFL and tumor were obtained via tissue digestion. Briefly, fresh tissue was cut into small pieces and digested with 0.5 mg/mL of collagenase (Sigma-Aldrich, St. Louis, MO) and 0.1 mg/mL of DNase I (Roche, Indianapolis, IN) for 30 minutes at 37°C. Cell

suspensions were filtered through cell strainers and mononuclear cells (MNCs) were obtained by Ficoll density gradient centrifugation. Viability was determined by trypan blue exclusion. Formalin-fixed, paraffin-embedded sections (6 μm) from liver tissues were used for immunohistochemistry. Deparaffinized sections were boiled for 10 minutes in Tris (10 mM)/ethylene

diamine tetraacetic acid (1 mM) (pH 9.0) buffer for antigen retrieval. The sections were labeled with 10 μg/mL of anti-FoxP3 antibody (clone 236A/E7; AbCAM, Cambridge, Adriamycin research buy UK). Endogenous peroxidase blockage and the secondary reagent used to detect the primary antibody were from the EnVision+ System-HRP kit (Dako, Denmark). Tissue sections were counterstained with hematoxylin. PBMCs and MNCs isolated from TFL or tumor were analyzed for expression of surface and intracellular markers using the following anti-human antibodies: anti-ICOS, anti-GITR, anti-Ki67, anti-CD25, anti-CTLA-4, anti-granzyme B, anti-Perforin, anti-CD8, anti-HLA-DR, anti-FoxP3, anti–TNF-α, anti-CD4, anti-CD3, anti-CD56, anti-CD45, and anti-CD25 (see Supporting Information for details). Cells were analyzed in a FACSCanto II system (BD Biosciences, San Diego, CA). Tumor lysates were generated from freshly dissected tumors by five cycles of freezing and Suplatast tosilate thawing in phosphate-buffered saline, followed by filtration (0.2 μm), and normal liver (NL) lysates were made by the same method from TFLT tissue. Myeloid dendritic cells (mDCs) were isolated from PBMCs by positive selection (BDCA-1 dendritic cell isolation kit, Miltenyi

Biotec, Germany). mDCs were cultured overnight with media or 10 μg/mL autologous tumor lysate (TL), NL, or cytomegalovirus (CMV) antigens (Microbix Biosystems, Mississauga, Ontario, Canada) in the presence of 10 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) (Miltenyi Biotec) and 0.1 μg/mL of polyinosinic:polycytidylic acid (InvivoGen, San Diego, CA). CD4+CD25− cells were isolated from PBMCs or TILs that were kept overnight at 4°C in medium supplemented with 10% fetal bovine serum, by magnetic sorting (Miltenyi Biotec). CD4+CD25− T cells were labeled with 0.1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen) and cocultured with autologous mDCs, pulsed with media, TL, NL, or CMV, at a ratio of 1:10 for 5 days in round-bottom 96-well plates with at least 5 × 104 CD4+CD25− T cells.