Abstract
BackgroundDendritic 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.MethodsShort 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.ResultsRIG-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-β1 than did reference cells. The tumors formed after injection of RIG-I-deficient HCC cells had higher TGF-β1 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-β1 antibody. TGF-β1-induced phosphorylation of Smad2 and Akt was enhanced in RIG-I-deficient HCC spheres, knockdown of AKT gene expression abolishing the augmentation of TGF-β1-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-β stimulation.ConclusionsOur results demonstrate that RIG-I deficiency in HCC cells induced their stemness, enhanced secretion and signaling of TGF-β1, tolerogenic TIDCs and less generation of DCs, and the results suggest involvement of TGF-β1 in those RIG-I deficiency-induced tolerogenic changes and involvement of CSCs in DC-mediated immunotolerance.
Highlights
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
The cultures containing DCs generated in the presence and absence of the conditioned medium (CM) of NCsh spheres or the CM of CRIG-Ish spheres were treated with mitomycin C and co-cultured with allogeneic CD3+ T cells
Among T cells co-cultured with CRIG-Ish spheres-CM-treated DCs, NCsh spheres-CM-treated DCs and CM-untreated DCs, T cells co-cultured with CM-untreated DCs exhibited the highest rate of proliferation, and T cells co-cultured with CRIG-Ish spheres-CM-treated DCs showed the lowest rate of proliferation, indicating Retinoic acid-inducible gene I (RIG-I) deficiency-induced immune tolerance (Fig. 3d)
Summary
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. Dendritic cells (DCs) are antigen-presenting cells that find, capture, process and present antigens on the cell surface along with appropriate costimulation molecules such as the B7 family, tumor necrosis factor family and intracellular adhesion molecules They activate and differentiate CD4+ and CD8+ T cells to develop an adaptive immune response. A recent study by Li et al has shown that retinoic acid-inducible gene-I (RIG-I), a pivotal cytoplasmic molecular pattern recognition receptor, is involved in the regulation of maintenance of leukemic stemness: RIG-I deficiency increases leukemic stemness [5].
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