The Study Of Tumor Infiltrating Lymphocytes Transfected With TRAIL And Interleukin-2 Gene In Renal Cell Carcinoma

Objectives. To investigate whether tumor infiltrating lymphocytes (TIL) transfected with Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) and interleukin-2 (IL-2) genes is capable of improving the potency and efficiency of propagation and cytotoxicity against renal cell carcinoma (RCC) cells in vitro.Methods. TRAIL and IL-2 cDNA were amplified by RT-PCR, and then was subcloned into the vector pMD18-T-simple. The amplified TRAIL and IL-2 cDNA fragment containing the entire coding sequence was confirmed by sequencing analysis respectively. The restriction enzymes were employed for digestion to verify the designed restriction enzymes sites in the primer. A mammal expression vector system was constructed by subcloned two genes into pBudCE4.1 in which two strong promoters CMV and EF-lα were employed. Three recombined vector pBudCE4.1 -IL-2, pBudCE4.1 -TRAIL and pBudCE4.1 -TRAIL-IL-2 were constructed. TILs and fresh RCC cells were derived from 10 patients with RCC undergoing radical nephrectomy. TIL was transfected by liposome-mediated gene transfection. The degree of cytokine mRNA expression was evaluated with Northern blot. Protein expression was determined with Western blot and ELISA. Cytotoxicity of TIL (effector cell, EC) against autologous RCC cells and human RCC cell line (target cell, TC) were examined by chromium release assay (EC : TC=20 : 1). Flow cytometric analyses were performed to determine the apoptosis of tumor cells. There is no significance between TIL/Parental and TIL/vector in all determined values.Results. High level expression of the human TRAIL and IL-2 genes stably transfected TIL cells were observed. Mean IL-2 production were 22.6±5.2, 507.7±52.4 and 549.0±74.0 ng/10~6cells/24h in the TIL/Parental, TIL/IL-2 andTIL/TRAIL+IL-2, respectively. The mean cytotoxicity (effector/target ratio 20:1) of TIL/Parental, TIL/IL-2, TIL/TRAIL and TIL/TRAIL+IL-2 against autologous RCC cells in percent cytolysis were 21.2±4.8%, 32.1±5.5%, 63.5±6.6%, and 78.1±9.63%, respectively. These four groups showed cytotoxic activities against allogeneic 786-0 RCC cells;the results were 9.8±3.5%, 12.3±3.4%, 24.1±4.9%, and 30.4±6.2%, respectively. The number of apoptosis cells was significantly more for autologous RCC cells than for 786-0 cells by TIL/TRAIL and TIL/TRAIL+IL-2 treatment, respectively.Conclusions. TIL/TRAIL+IL-2 and TIL/IL-2 genes were expanded by autocrine IL-2. TIL/TRAIL+IL-2 and TIL/TRAIL showed significant cytotoxicity induced by TRAIL. TIL including parental TIL and transfected TIL demonstrates a potent cytotoxicity against RCC cells with remarkable selectivity. Autologous RCC cells seemed more sensitive than allogeneic RCC cells. TIL transfected with TRAIL enhanced the potence induce the apopotosis of RCC.