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. 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. 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-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. Our 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.

A reduced number and/or reduced activity of natural killer (NK) cells, which are important for defense against a variety of cancers and viral infections, occur under various stress conditions and in patients with various diseases. In this article, we report that the 30% to 50% ethanol precipitate of oyster extract (EPOE50) dose-dependently enhanced the activity of mouse spleen NK cells in vitro and in vivo. The activity of EPOE50 was eluted with a molecular weight of about 2000 by gel filtration and was inactivated by periodate but not by proteinase K. The activity of highly purified NK cells was also augmented by EPOE50 but not by oligodeoxyribonucleotide 1585, which mimics bacterial DNA. Administration of EPOE50 to mice stimulated splenic NK cell activity without a change in splenic NK cell populations. Although the proliferation of B16 tumor cells in vitro was slightly stimulated by EPOE50, the growth of B16 melanoma in vivo was dose-dependently suppressed by administration of EPOE50. Taken together, our results indicate that EPOE50 augmented NK cell activity and that its administration to mice inhibited tumor growth presumably through the activation of NK cells and also suggest that the active substance is a sugar-containing oligomer or polymer and is not of bacterial origin.

Title: Effectiveness of and Immune Responses to SARS-CoV-2 mRNA Vaccines and Their Mechanisms | Keywords: COVID-19, SARS-CoV-2, mRNA vaccine, immune response | Author: Eiichi Gohda