HCC tissue sections were stained with rat antihuman Tim-3 (R&D), and then with HRP-conjugated goat antirat IgG (1/500, Invitrogen). Visualization was achieved with ABC-Elite Reagent (Sigma). The sections were counterstained with Mayer’s hematoxylin (Sigma). The nuclei were stained with 1% ammonium hydroxide. The numbers of Tim-3+ cells were counted in five fields at ×400 magnification. Real-time PCR was performed as described.14, 19 Specific primers are listed in Supporting Table 1. Transwell chambers with a 0.4 μm pore membrane (Corning-Costar) were used. Acalabrutinib CD14+ cells (5 × 105/mL) from the blood of healthy donors or normal KCs from relatively normal liver tissues

with hepatic hemangiomas were plated to the lower chambers. T cells isolated from HCC tissues or adjacent tissues were added to the upper chamber and cultured with interferon (IFN)-γ (400 U/mL) for 48 hours. CD14+ cells were collected and galectin-9 expression was determined by flow cytometry. Antihuman IFN-γ mAb (500 ng/mL, R&D) was added to the culture as indicated. Comparisons were made using the Wilcoxon test. Survival curves were compared by the Kaplan-Meier method and the log-rank test, and survival was measured in months from resection to the last review. The log-rank test was applied to compare the groups. Multivariate analysis of prognostic factors for survival data was performed using the Cox proportional hazards model. Differences in values at P < 0.05 were

considered significant. RG 7204 All analyses were done using SPSS v12.0 software. To study the functional relevance of galectin-9 in patients with HCC, we examined the expression of galectin-9 on lin−CD45− HCC cells and different immune cell populations including T cells, HLA-DR+CD14+ KCs, lin−HLA−DR+CD4+CD11c+ myeloid dendritic cells (mDCs), and lin−HLA−DR+CD4+CD123+ plasmacytoid dendritic cells (pDCs), in paired HBV-associated HCC tissues and surrounding nontumor adjacent tissues. Flow cytometry analysis revealed that tumor cells and T cells expressed minimal galectin-9 (<4%), pDCs and mDCs expressed moderate levels of galectin-9 (10%), and KCs expressed the highest

levels of galectin-9 in HCC (Fig. 1A). Next we compared the expression of galectin-9 on KCs in HCC tissues and adjacent tissues from both HBV-positive and -negative patients. In HBV-positive patients the percentage of galectin-9+ KCs was higher Edoxaban in tumor tissues than in adjacent tissues (46.8 ± 3.9% versus 10.7 ± 2.3%) (Fig. 1B). However, in HBV-negative patients (Fig. 1B) the levels of galectin-9 expression on KCs were negligible (<0.5%) in both HCC and adjacent tissues. Immune fluorescence staining confirmed that there were higher numbers of galectin-9+CD68+ KCs in HCC tumor tissues (38 ± 13%) than in adjacent nontumor tissues (11 ± 5%) (Fig. 1C). The data indicate that KCs are the primary galectin-9-expressing APC subset in HBV-associated HCC. Next we investigated why KCs express high levels of galectin-9 in HCC.