NK cells (also known as natural killer cells) play important functions in innate immune system and are responsible for lysing cells such as transformed, computer virus infected cells or stressed cells without prior sensitization or MHC class restriction

NK cells (also known as natural killer cells) play important functions in innate immune system and are responsible for lysing cells such as transformed, computer virus infected cells or stressed cells without prior sensitization or MHC class restriction. shown that this inflammatory microenvironment of HCC contributes to antitumor immunosuppression. Transmission transducer and activator of transcription 3 (STAT3) functions a pivotal role in determining and maintaining a procarcinogenic inflammatory microenvironment during tumor progression [[4], [5], [6], [7]], and in HCC, the interleukin (IL)-6/STAT3 pathway has important functions Deoxycholic acid in tumor progression [8,9]. In tumor microenvironment, IL-6/STAT3 pathway plays to promote the survival, proliferation, and invasiveness of tumor cells, while intensively suppressing the antitumor immunity [10]. Recent studies have shown that anti-inflammatory strategies hold promise in malignancy treatment [11]. We and others have shown that inhibition of aberrant STAT3 activation or deletion of STAT3 in mice suppresses tumor progression by improving antitumor immune responses [[12], [13], [14]]. Besides, STAT signaling also plays important functions in regulation of innate immunity of human population especially in people with Deoxycholic acid tumors [15]. Furthermore, many clinical and/or preclinical studies have Deoxycholic acid also shown that this anti-inflammatory agents used to target cancer-related inflammation enhance the effects of immunotherapies and suppress malignancy progression [16]. Liraglutide is a glucagon-like peptide-1 (GLP-1) mimetic with 97% structural homology to GLP-1 [17] that promotes insulin secretion [18] and reduces post-prandial glucose levels [19] in people with type 2 diabetes. Interestingly, liraglutide has also been shown to have anti-inflammatory activity in non-alcoholic steatohepatitis [20]. Furthermore, liraglutide pretreatment was shown to significantly reduce IL-1 levels and effectively inhibit the formation of the NLRP3 inflammasome [21]. In line with these results, GLP-1 was observed to markedly inhibit ovalbumin-induced airway inflammation and NF-B p65 activation [22]. Therefore, we hypothesized that liraglutide may possess antitumor efficacy by modulating the inflammatory microenvironment of HCC. In our experiment, we showed liraglutide enhances the antitumor activity of NK cells by inhibiting IL-6/STAT3 signaling. NK cells (also known as natural killer cells) play important roles in innate immune system and are responsible for lysing cells such as transformed, virus infected cells or stressed cells without prior sensitization or MHC class restriction. In human body, NK cells are identified as CD3?CD16+CD56+ and NKp46 (natural cytotoxicity receptor) Deoxycholic acid positive lymphocytes [23]. In this research, we studied the therapeutic role of liraglutide in manipulating the antitumor immune responses in HCC and attempted to elucidate the underlying mechanism. Materials and methods Cell lines The hepatocellular carcinoma cell lines HCC-LM3 and Hepa1-6 were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in complete DMEM supplemented with 10% fetal bovine serum, 100?units/mL penicillin, 100?g/mL streptomycin and 2?mM l-glutamine (all from Life Technology, NY, USA) and kept at 37?C in a humidified atmosphere containing 5% CO2. Generation of NKT cells Human peripheral blood (50?mL) was obtained from healthy male volunteers, and all volunteers have signed the informed consent. Human PBMCs were isolated freshly via density gradient centrifugation using Ficoll (Hao Yang Biological Products Technology, Tianjin, China) as described elsewhere [24,25]. Cells were then washed with PBS and propagated at 1??106 cells/mL in GT-T551 (Takara Bio, Shiga, Japan) containing 2000?IU/mL recombinant human IFN- (Chemo Wanbang Biopharma, Shanghai, China), 10?mg/mL OK432 (Shanghai Chugai Pharmaceutical, Chome, Japan), 1% penicillin-streptomycin and 2% heat-inactivated autologous plasma for 24?h. After that, cells were transferred to a cell culture flask with 5?g/mL anti-CD3 antibody coated (T&L Biological Technology, Beijing, China). Then, GT-T551 medium containing 700?IU/mL recombinant human IL-2 was added (BD Biosciences, CA, USA). After 14?days, cells were detected by flow cytometry, collected and kept at ?80?C for further use. The experiment was approved by the Ethics Committee of Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, China. Flow cytometry The quality and phenotype of NKT cells were determined by flow cytometry. Briefly, Akt1s1 cells were harvested and cultured for 30?min at 4?C using four fluorescence-conjugated antibodies: anti-CD4-APC, anti-CD3-FITC, anti-NKG2D-APC and anti-CD8-PE (all from BD Biosciences, CA, USA). Then, the NKT cells were detected using a FACS-Caliber System (from BD Biosciences, CA, USA) before washed twice with PBS. The results were analyzed using FlowJo software (Version 7.6.5, Tree Star Inc., Deoxycholic acid OR, USA). Animals Male C57BL/6J mice (6C8?weeks old) were purchased from the Model Animal Research Center of Nanjing University. All mice were contained in an idea microenvironment (temperature: 23??2?C, humidity: 60??10%, and a 12/12?h light/dark cycle) with free access to food and.