Induction of tolerogenic dendritic cells by activated TGF-ß/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cells.

BACKGROUND: Dendritic cells (DCs) alter their role from being immunostimulatory to immunosuppressive at advanced stages of tumor progression, but the influence of cancer stem cells (CSCs) and their secreted factors on generation and phenotypic change of DCs is unknown. Retinoic acid-inducible gene I (RIG-I) plays a role in regulation of other cellular processes including leukemic stemness besides its antiviral function. METHODS: Short hairpin RNA-mediated gene silencing was employed to generate stable RIG-I-knocked-down human hepatocellular carcinoma (HCC) cell lines. Expression levels of genes and proteins in spheres of those HCC cells were determined by quantitative real-time PCR and Western bot, respectively. Levels of secreted cytokines were measured by ELISA. The surface molecule expression levels of DCs were analyzed using flow cytometry. The ability of DCs to induce proliferation of T cells was assessed by a mixed lymphocyte reaction (MLR) assay. RESULTS: RIG-I-knocked-down HCC cells showed upregulated expression of stem cell marker genes, enhanced secretion of factors suppressing in vitro generation of DCs into the conditioned medium (CM), and induction of a phenotype of tumor-infiltrating DCs (TIDCs) with low levels of DC markers in their tumors in nude mice. Those DCs and TIDCs showed reduced MLR, indicating RIG-I deficiency-induced immunotolerance. The RIG-I-deficient HCC cells secreted more TGF-ß1 than did reference cells. The tumors formed after injection of RIG-I-deficient HCC cells had higher TGF-ß1 contents than did tumors derived from control cells. DC generation and MLR suppressed by the CM of RIG-I-deficient HCC cells were restored by an anti-TGF-ß1 antibody. TGF-ß1-induced phosphorylation of Smad2 and Akt was enhanced in RIG-I-deficient HCC spheres, knockdown of AKT gene expression abolishing the augmentation of TGF-ß1-induced Smad2 phosphorylation. Akt and p-Akt were co-immunoprecipitated with Smad2 in cytoplasmic proteins of RIG-I-deficient spheres but not in those of control spheres, the amounts of co-immunoprecipitated Akt and p-Akt being increased by TGF-ß stimulation. CONCLUSIONS: Our results demonstrate that RIG-I deficiency in HCC cells induced their stemness, enhanced secretion and signaling of TGF-ß1, tolerogenic TIDCs and less generation of DCs, and the results suggest involvement of TGF-ß1 in those RIG-I deficiency-induced tolerogenic changes and involvement of CSCs in DC-mediated immunotolerance.

Restoration of stemness-high tumor cell-mediated suppression of murine dendritic cell activity and inhibition of tumor growth by low molecular weight oyster polysaccharide.

Dendritic cells (DCs) play key regulatory roles in tumor immunity: increased activity of DCs infiltrating tumor tissues leads to enhancement of tumor immunity. Functions of DCs are also modulated by tumor cell-derived factors. Here, we investigated the effects of low molecular weight oyster polysaccharide (LMW-OPS) on differentiation and function of bone marrow-derived DCs (BMDCs) exposed to a conditioned medium (CM) obtained from spheres of stemness-high colorectal cancer cell lines CMT93 and CT26. The CM containing a detectable level of TGF-ß1 was found to down-regulate the surface expression of major histocompatibility complex class II of BMDCs and to inhibit the potency of BMDCs to stimulate T cells. Those suppressions were partly restored and completely restored by addition of anti-TGF-ß1 and LMW-OPS, respectively. Production of IFN-γ during allogeneic T cell responses was inhibited by the CM, whereas production of TGF-ß1 was augmented by the CM. The IFN-γ profile was also reversed by addition of LMW-OPS. Nuclear translocation of ß-catenin, but not that of NF-κB p65, was induced by TGF-ß1. NF-κB p65 nuclear translocation, but not ß-catenin nuclear translocation, was induced by LMW-OPS. Intraperitoneal injection of LMW-OPS significantly suppressed tumor growth in syngeneic tumor models using CMT93 and CT26 sphere cells, whereas it had no inhibitory effect on the proliferation of either cell line. The results demonstrated that LMW-OPS relieved stemness-high tumor cell-mediated suppression of BMDC function and indicated the in vivo anti-tumor activity of LMW-OPS in which re-stimulation of the activity of DCs infiltrating tumor tissues is presumed to be involved.

Activation of dendritic cells by low molecular weight oyster polysaccharides.

Dendritic cells play a primary role in antigen presentation to CD4+ T cells, which initiate acquired immune responses. Therefore, determining positive modulators of dendritic cell activation to improve therapeutic approaches for cancer treatment might be useful. We here investigated the effects of low molecular weight oyster polysaccharides (LMW-OPS) on bone marrow-derived dendritic cells (BMDCs) obtained from mice. LMW-OPS increased the surface expression of major histocompatibility complex class II (MHC-II), CD40 and CD86 in BMDCs and induced the secretion of tumour necrosis factor (TNF)-α and interleukin (IL)-12, which were significantly decreased in the BMDCs derived from MyD88-/- mice but not from the lipopolysaccharide-resistant C3H/HeJ mice. BMDCs treated with LMW-OPS augmented allogeneic CD4+ T cell expansion and enhanced secretion of IL-2 and interferon (IFN)-γ but not IL-4. LMW-OPS induced significant increases in ERK and p38 MAPK phosphorylation, but not c-Jun N-terminal kinase (JNK) phosphorylation, in BMDCs. Our results indicate that, in part, LMW-OPS can induce maturation of BMDCs in a MyD88-dependent and Toll-like receptor (TLR) 4-independent manner. LMW-OPS may enhance acquired immunity by modulating the function of dendritic cells.