Full Length Research Paper

Regression of mouse-derived renal cancer by adoptive transfer of tumor-reactive RNAi-induced TGF-beta-insensitive CD8⁺ T cells

Bin Song¹*, Wei-jun Qin², Zeng-yue Yang¹, Ting-yi Bao¹, Xia-Yang³, Fu-li Wang², Geng Zhang², An-gang Yang⁴ and He Wang²

¹Department of Urology, Tangdu Hospital, Fourth Military Medical University, Xin Si Road, Xi'an, 710038, China.

²Department of Urology, Xijing Hospital, Fourth Military Medical University, Chang Le West Road 15, Xi'an, 710032, China.

³Department of Division of Medical Service, Tangdu Hospital, Fourth Military Medical University, Xin Si Road, Xi'an, 710038, China.

⁴Department of Immunology, Fourth Military Medical University, Chang Le West Road 17, Xi'an, 710032, China.

*Corresponding author. E-mail: songbin006@gmail.com,  hewang.fmmu@gmail.com.  Tel: +86-29-84777728. Fax: +86-29-84775321.

Abbreviations: 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.

Accepted 2 August, 2010

   Abstract

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.