For in vitro experiments, 38 paired fresh tissues were used from HCC patients,

including 30 HBV+ cases and eight HBV− alcoholic cases in different experiments. Fresh HCC tissues and surrounding nontumor adjacent liver tissues (at least 3 cm distant from the tumor site) were used Fulvestrant clinical trial for the isolation of tumor- and nontumor-infiltrating leukocytes. For survival analysis, we followed 99 HBV-associated HCC patients after surgical resection from January 2007 to April 2010 (Table 1). The research was approved by the Institutional Review Board of Tongji Medical College of Huazhong University of Science and Technology. Both written and oral consent was obtained before samples were collected. Immune cells were obtained from peripheral blood and fresh liver tissues as described.19 CD14+ tumor-associated Kupffer cells (KCs) and Tim-3+CD4+ T cells were isolated with paramagnetic beads (StemCell Technology, Canada) and sorted.

Cell purity was >90% as confirmed by flow cytometry (LSR II, Becton Dickinson). Immune cells were stained extracellularly with fluorochrome-conjugate-specific antibodies against human antibodies, then fixed and permeabilized with Perm/Fix solution (eBioscience), and stained for intracellular cytokines and Ki67 (eBioscience). Tim-3+CD4+ T cells (5 × 105/ml) were cocultured with CD14+ KCs (105/mL) from the same HCC tissue from six patients for 5 days in the presence of antihuman CD3 (2.5 μg/mL, BD Biosciences) and antihuman CD28 (1.25 μg/mL) or with autologous HCC (105/mL). Neutralizing monoclonal antibody (mAb) Tryptophan synthase against Pifithrin-�� human Tim-3 (10 μg/mL, Biolegend) or isotype controls were added to the culture. The resultant cells were collected for flow cytometry analysis or for ELISPOT assay with ImmuneSpot analyzer (Cellular Technology).

Carboxyfluorescein succinimidyl ester (CFSE)-labeled Tim-3+CD4+ T cells were incubated with CD14+ KCs from the same HCC tissue from six patients for 5 days. Cell division was determined based on CFSE dilution by flow cytometry analysis. Frozen tissue sections were stained with primary antibodies, rat monoclonal antihuman Tim-3 (clone: 344823, 1/200, IgG2a, R&D Systems), mouse antihuman CD4 (Clone: RPA-T4, 1/500, IgG1, eBioscience), mouse antihuman galectin (clone: 9M1-3, 1/500, IgG1, Biolegend), and CD68 (clone: Y1/82A, 1/500, IgG2b, eBioscience), and subsequently stained with secondary antibodies, Alexa Fluor 568-conjugated goat antirat IgG2a, Alexa Fluor 488-conjugated goat antimouse IgG1, and Alexa Fluor 568-conjugated goat antimouse IgG2b (all 2 μg/mL, Invitrogen). Hoechst 33342 (Invitrogen) was used for nuclear staining. Images were acquired by fluorescence microscope and positive cells were quantified by ImagePro Plus software (Media Cybernetics, Bethesda, MD) and expressed as the mean of the percentage of positive cells ± standard error of the mean (SEM) in five high-powered fields.