Transforming growth factor beta (TGF-beta) is a potent immunosuppressant. The present study was conducted to develop a treatment strategy through adoptive transfer of tumor-reactive RNAi-induced TGF-beta-insensitive CD8+ T cells. BALB/c mice were primed with irradiated Renca cells. CD8+ T cells were isolated from the spleen of primed animals, expanded ex vivo and were rendered TGF-beta-insensitive by infecting with a retrovirus containing shRNA to mouse TGF-beta type II receptor gene (MSCV-shRNA-T). Control CD8+ T cells consist of those infected with retroviruses containing shRNA to non specific gene (MSCV-shRNA-N) and naive CD8+ T cells. The effect of all groups of CD8+ T cells on Renca cells were analyzed by semi-quantitative RT-PCR, Western-blot, in vitro and in vivo assay. MSCV-shRNA-T group of CD8+ T cells were resistant to the antiproliferative effect of exogenous TGF-beta, while control groups were not. Results of Western blot showed the Smad pathway was disrupted in MSCV-shRNA-T group, which confirmed the blockade of the signal transduction pathway. In vitro cytotoxic assay revealed that these tumor-reactive, TGF-beta-insensitive CD8+ T cells killed Renca cells specifically and strongly. Adoptive transfer of these MSCV-shRNA-T CD8+ T cells to BALB/c tumor-bearing mice showed strong tumor-specific cytotoxic T lymphocyte responses and antitumor immunity against Renca renal cancer. Based on these results, we predict that adoptive transfer of tumor-reactive RNAi-induced TGF-beta-insensitive CD8+ T cells may be effective to renal cancer therapy.
Key words: Transforming growth factor beta, adoptive transfer, RNA interference, renal cancer, renca cells, immunotherapy.
CM, Complete medium; CTL, cytotoxic thymus lymphocyte; FBS,fetal bovine serum; GFP, green fluorescent protein; TGF-ß, transforming growth factor beta; Renca, mouse-derived renal cancer; TßRI, type I TGF-ß receptor;TßRII, type II TGF-ß receptor; TßRIII, type III TGF-ß receptor; RNAi, RNA interference; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; MHC, major histocompatibility complex; MSCV, murine stem cell virus; DMEM, dulbecco's modification of eagle's medium; RT-PCR, reverse transcription-polymerase chain reaction.
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