At day 5, DCs were stained with different fluorescent-conjugated antibodies including anti-CD14 FITC (Clone 18D11, Immunotools) for monocyte marker, anti-CD11c APC (Clone BU15, Immunotools) for DC marker, anti-CD86 PE (Clone IT2.2, eBioscience), anti-CD40 FITC (Clone HI40a Immunotools), and anti-HLA-DR FITC (MEM12, Immunotools) for co-stimulatory molecules, and Bicyclol anti-CD83 PE (Clone HB15e; eBioscience) for DC maturation marker, at 4C for Bicyclol 30 minutes. membrane of DCs, as shown by Western Rabbit Polyclonal to EIF3K blot analysis and immunofluorescence staining, whereas the IL-10 and TGF- ligands were detected in the culture supernatants of DCs and cholangiocarcinoma (CCA) cell line, respectively. Inhibition of the IL-10 and TGF- receptors on DCs by specific neutralizing antibodies significantly increased level of IFN- and enhanced cytolytic activity of the DC-activated effector T-cells against CCA cell line. These results indicate that this IL-10 and TGF- receptors are the targets for inhibition to increase DC functions and enhance cytolytic activity of the DC-activated effector T-cells against CCA cells. Thus, inhibition of the IL-10 and TGF- receptors on DCs is crucial in the preparation of DC-activated effector T cells for adoptive T-cell therapy. found that expression of TGF- in renal adenocarcinoma reduced the efficacy of DC-based immunotherapy in Bicyclol mice model.9 Furthermore, the study by Dumitriu IE showed that lung carcinoma cell-culture supernatant treated DCs reduced expression of CD86 and production of IL-12 and TNF-.10 These results indicated that immunosuppressive cytokines are important factors that can induce tolerogenic DC. Cholangiocarcinoma (CCA) is usually a malignancy of bile duct epithelial cells. This cancer has highest incidence in the population living in the Northeastern a part of Thailand where there is usually highly prevalence of liver fluke (study exhibited that tumor-derived factors in the culture supernatant from intrahepatic CCA cell lines could induce macrophage cell line polarization toward tumor-associated macrophages (TAMs) that had ability to produce immunosuppressive factors such as IL-10, TGF-, VEGF-A.12 The patients with CCA showed positive TGF-1 expression that significantly correlated with lymph node metastasis, distant metastasis, and tumor recurrence.13 Moreover, the vaccination of synthesized Wilms tumor 1 (WT1) and/or mucin 1 (MUC1) peptides in the patients with advanced stage of CCA showed positive responses with minimal toxicity.14 However, clinical outcomes of this vaccination were unsatisfactory.14 Since CCA can produce immunosuppressive cytokines to impair DC function, we hypothesize that inhibition of these cytokines or their receptors enhance the DC function to mediate anti-tumor immunity. To test this hypothesis, we used specific neutralizing antibodies to inhibit IL-10 and TGF- receptors on DCs and examined DC functions. Herein, we report our finding that inhibition of the IL-10 and TGF- receptors on DCs by specific neutralizing antibodies significantly improved DC function to enhance cytolytic activity of DC-activated effector T-cells against CCA cells. Results Generation of dendritic cells DCs were generated from human monocytes isolated from PBMCs by stimulation with recombinant cytokines. The percentage of CD11c?CD14+ cells, representing monocyte population, was decreased and differentiated into CD11c+CD14? cells, representing monocyte-derived DC population at day 5 (Fig.?1A-B). The DC morphology after staining with FITC-conjugated anti-human HLA-DR antibody was observed under a fluorescence microscopy. The results revealed that immature DCs showed round Bicyclol shape, smaller in size than mature DCs, whereas mature DCs showed the morphology of roughness, cytoplasmic projections, and ruffles around the cell surface with protrusions of dendrites. In addition, HLA-DR was found to be up-regulated in mature DCs than immature DCs, representing the maturation status of DCs (Fig.?1C). Immunophenotypes of DCs were further characterized by staining with antibodies specific to cell surface markers on DCs and then analyzed by flow cytometry (Fig.?1D). The results of immunophenotypic analysis revealed that CD11c which is a DC marker was highly up-regulated in mature DCs (MFI 132) compared with immature DCs (MFI 37.9), while CD14 which is a monocyte marker was down-regulated in mature DCs. The expression of DC maturation marker, CD83, was increased in mature DCs (MFI 15.5) as compared with immature DCs (4.79). The HLA-DR, CD86, and CD40, which are important for T-cell activation, were moderately increased in mature DCs (MFI 76.5, 272, 342) as compared with immature.
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