In a recent study,21 we observed that Th17 cells were highly enriched in HCCs and their levels

were positively correlated with microvessel density in tissues and poor survival in HCC patients. In contrast to the classical Th17 cells that buy Forskolin hardly express interferon (IFN)-γ, almost half of the IL-17-producing CD4+ T cells we isolated from HCC tissues were able to simultaneously produce IFN-γ, suggesting that the tumor microenvironment can profoundly determine the phenotype of such cells. Inasmuch as monocytes/Mψ represent an abundant population of antigen-presenting cells (APCs) in solid tumors and their density is inversely associated with the prognosis in HCC,8, 10 we investigated whether monocytes/Mψ can regulate Th17 and, if so, how they exert that influence, paying particular attention to the tissue microlocalization and phenotype of these cells in HCC. Ab, antibody; APCs, antigen-presenting cells; CCM, conditioned medium from control (untreated) monocytes; HCC, hepatocellular carcinoma; IFN, interferon; IL, interleukin; Mψ, macrophage(s); TAM, tumor-associated Mψ; TCM, conditioned medium from TSN-exposed

monocytes; TGF, transforming growth factor; Th, T helper; TIL, tumor-infiltrating leukocyte; TNF, tumor necrosis factor; TSN, tumor culture supernatant. Detailed information about the patients and specimens is described in the Supporting Materials and Methods and Supporting Table 1. Peripheral leukocytes were isolated by Ficoll density gradient centrifugation.8, 22 Isolation of tumor-infiltrating leukocytes (TILs) are detailed in the Supporting Materials and Methods. Detailed information about cell lines and

preparation of TSNs is described Lck in the Supporting selleck kinase inhibitor Materials and Methods. Monocytes were selected from peripheral blood mononuclear cells using anti-CD14 magnetic beads (Miltenyi Biotec). To generate conditioned media, monocytes were left untreated or cultured for 1 hour with 20% TSN from HepG2 cells and then washed and cultured in RPMI 1640 containing 10% human AB serum for 16 hours. Thereafter, the supernatants were harvested, centrifuged, and stored in aliquots at −80°C. Concentrations of cytokines were determined using ELISA kits (eBioscience, San Diego, CA). In 2-day incubation, purified CD3 T cells, naive T cells, and memory T cells (Miltenyi Biotec) were left untreated or were cocultured with autologous monocytes or Mψ or were exposed to 50% conditioned medium or medium supplemented with recombinant IL-6, IL-23, and/or IL-1β (Peprotech) in the presence of 2 μg/mL anti-CD3 and 1 μg/mL anti-CD28. Thereafter, cells were washed and maintained in RPMI medium supplemented with 20 IU/mL IL-2 for indicated times with conditioned medium or different cytokines. In some experiments, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) or pretreated with a neutralizing Ab against IL-6, IL-23, or IL-1β, or a control immunoglobulin G (IgG) (R&D Systems), and subsequently exposed to conditioned media.