Saiming Chen1, Zhiqun Li1, Limin Zhou2, Yunxia Zhang2. 1. Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Hainan Medical College. 2. Scientific Experimental Center of Hainan Medical College, Haikou 570102, China.
Abstract
OBJECTIVE: To investigate the effect of sputum ubiquitin ligase (Cbl-b) gene known-down on the cytotoxicity of H9 T lymphocytes against human laryngeal squamous cancer Hep-2 cells and explore the underlying mechanism. METHODS: CD4+ T lymphocytes isolated from 12 patients with laryngeal squamous carcinoma and 12 healthy individuals were examined for Cbl-b mRNA expressions using RT-PCR. H9 T lymphocytes cultured in 96-well plates were transfected with Cbl-b siRNA via liposomes followed by treatment with an anti-IL-2 monoclonal antibody, with H9 T lymphocytes transfected with a scrambled sequence as the negative control. The expressions of Cbl-b mRNA and protein in the cells were detected using real-time fluorescent quantitative PCR and Western blotting, respectively. The killing effect of the treated T lymphocytes against Hep-2 cells was assessed using the cell counting kit (CCK-8). The positive expression rates of CD69 and CD25 on the surface of H9 T lymphocytes were determined using flow cytometry, and the levels of interleukin-2 (IL-2) and interferon-gamma (INF-γ) in the culture supernatants of H9 T lymphocytes were detected with ELISA. RESULTS: The CD4+ T lymphocytes from patients with laryngeal squamous carcinoma showed significantly increased Cbl-b mRNA level compared with those from healthy individuals (P < 0.05). Transfection of H9 T lymphocytes with Cbl-b siRNA significantly reduced the expression levels of Cbl-b mRNA and protein (P < 0.05), which were not significantly affected by subsequent treatment of the cells with the anti-IL-2 antibody (P>0.05). At different target-effector ratios, the Cbl-b siRNA-transfected cells showed significantly higher Hep-2 cell killing rates and higher positivity rates of CD69 and CD25 expressions than the blank and negative control cells and the cells with both Cbl-b siRNA transfection and anti-IL-2 treatment (P < 0.05). Cbl-b silencing in H9 T lymphocytes resulted in significantly increased levels of IL-2 and INF-γ in the supernatant as compared with those in the blank and negative control groups (P < 0.05). CONCLUSIONS: Cbl-b gene silencing effectively enhances the killing effect of H9 T lymphocytes against Hep-2 cells in vitro probably as the result of enhanced IL-2 secretion and T lymphocyte activation.
OBJECTIVE: To investigate the effect of sputum ubiquitin ligase (Cbl-b) gene known-down on the cytotoxicity of H9 T lymphocytes against humanlaryngeal squamous cancer Hep-2 cells and explore the underlying mechanism. METHODS: CD4+ T lymphocytes isolated from 12 patients with laryngeal squamous carcinoma and 12 healthy individuals were examined for Cbl-b mRNA expressions using RT-PCR. H9 T lymphocytes cultured in 96-well plates were transfected with Cbl-b siRNA via liposomes followed by treatment with an anti-IL-2 monoclonal antibody, with H9 T lymphocytes transfected with a scrambled sequence as the negative control. The expressions of Cbl-b mRNA and protein in the cells were detected using real-time fluorescent quantitative PCR and Western blotting, respectively. The killing effect of the treated T lymphocytes against Hep-2 cells was assessed using the cell counting kit (CCK-8). The positive expression rates of CD69 and CD25 on the surface of H9 T lymphocytes were determined using flow cytometry, and the levels of interleukin-2 (IL-2) and interferon-gamma (INF-γ) in the culture supernatants of H9 T lymphocytes were detected with ELISA. RESULTS: The CD4+ T lymphocytes from patients with laryngeal squamous carcinoma showed significantly increased Cbl-b mRNA level compared with those from healthy individuals (P < 0.05). Transfection of H9 T lymphocytes with Cbl-b siRNA significantly reduced the expression levels of Cbl-b mRNA and protein (P < 0.05), which were not significantly affected by subsequent treatment of the cells with the anti-IL-2 antibody (P>0.05). At different target-effector ratios, the Cbl-b siRNA-transfected cells showed significantly higher Hep-2 cell killing rates and higher positivity rates of CD69 and CD25 expressions than the blank and negative control cells and the cells with both Cbl-b siRNA transfection and anti-IL-2 treatment (P < 0.05). Cbl-b silencing in H9 T lymphocytes resulted in significantly increased levels of IL-2 and INF-γ in the supernatant as compared with those in the blank and negative control groups (P < 0.05). CONCLUSIONS:Cbl-b gene silencing effectively enhances the killing effect of H9 T lymphocytes against Hep-2 cells in vitro probably as the result of enhanced IL-2 secretion and T lymphocyte activation.
Authors: Leila Ben Merzoug; Solenne Marie; Naoko Satoh-Takayama; Sarah Lesjean; Marcello Albanesi; Hervé Luche; Hans Jörg Fehling; James P Di Santo; Christian A J Vosshenrich Journal: Eur J Immunol Date: 2014-09-19 Impact factor: 5.532
Authors: Raquel Gomez-Eerland; Bastiaan Nuijen; Bianca Heemskerk; Nienke van Rooij; Joost H van den Berg; Jos H Beijnen; Wolfgang Uckert; Pia Kvistborg; Ton N Schumacher; John B A G Haanen; Annelies Jorritsma Journal: Hum Gene Ther Methods Date: 2014-09-22 Impact factor: 2.396