The Killing Effect Of Tumor-reactive RNAi-induced TGF-β-insensitive CD8+T Cells To Mouse Renal Cancer Cell Line In Vitro And In Vivo

[Background]Renal cell carcinoma (RCC) is the most common malignancy in urogenital system. Although the incidence of RCC occupied only 3% of systemic malignant tumors, the incidence of RCC in the whole world increased 2% per year recently, and every year more than 100,000 people died of RCC. In our country, the incidence of RCC, which is inferior to the incidence of bladder cancinoma, occupys the second place in urogenital tumors. In recent years, there has been a ascending tendency of the RCC incidence and RCC is one of the most important tumors which threaten the people's health. Most RCCs are congenitally multidrug resistant and insensitive to chemotherapy and radiotherapy. In addition to that surgical treatment of early stage RCCs can achieve a better therapeutic efficacy, there are no specific treatment methods to most advanced RCCs nowadays. Hower, about 50% patients with RCCs are diagnosed as intermediate or advanced stage of RCC for the first visit. In about 40% patients, metastasis and recurrence happened. The prognosis of metastatic RCCs is bad. The mean survival time of patients with metastatic RCCs was less than one year and the three-year survival rate of the patients was only less than 5%. Therefore, to find a effective and specific treatment method to RCCs is the exigent problem to resolve in the clinic therapy of urology.The development of immunogene therapy has brought the new hope to the people. Both the selection of target gene and the effector cell are the key points of immunogene therapy. Transforming growth factorβ(TGF-β) is a multifunctional cytokine. It is important to cell proliferation, differentiation, apoptosis and development of lots of tissues. TGF-βis a potent immunosuppressant. The overproduction of TGF-βby tumor cells and the resulting inhibition of immune effector cells (T cell, B cell, macrophage, etc) in proliferation and differentiation may lead to tumor evasion from the host immune surveillance and tumor progression. Three types of TGF-βreceptors with high homology have been identified: typeⅠ(TβR-Ⅰ, 55KD), typeⅡ(TβR-Ⅱ, 75KD), and typeⅢ(TβR-Ⅲ, 280KD). They perform the signal transduction via the transmembrane receptor compounds. TβR-Ⅰand TβR-Ⅱexhibit serine/threonine kinase activity in their intracellular domains. TβR-Ⅲfunctions by binding TGF-βand then transferring it to its signaling receptors, the typeⅠandⅡreceptors. The current understanding shows that TGF-βfirst binds to TβR-Ⅱwith high affinity, which initiates intracellular signaling by phosphorylating several transcription factors and regulates the cell biological behaviour.It can be seen that TβR-Ⅱis the key molecule in TGF-βsignaling and CD8+ T cell is one of the effctor cells with killing effect in tumor immunity. We hypothesized that based on the premise that CD8+ T cells were tumor-reactive, knock-out of TβR-Ⅱon these CD8+ T cells via RNAi to make these tumor-reactive CD8+ T cells TGF-β-insensitive would resist the inhibition effect of TGF-βon proliferation and differentiation of CD8+ T cells, so that the killing effect of CD8+ T cells on tumor cells would be improved. This new strategy of immunogene therapy is expected to be provided in clinic for the treatment of RCCs.[Objective]To construct a retrovirus vector which contains the shRNA to mouse TβR-Ⅱgene. To product tumor-reactive TGF-β-insensitive CD8+ T cells by knocking down TβR-Ⅱon tumor-reactive CD8+ T cell surface via MSCV-incuced RNAi. To observe the killing effect of these CD8+ T cells to mouse renal cancer Renca cell line in vitro and in vivo.[Materials and methods]1 Selection of siRNAs and construction of MSCV-shRNAs retrovirus vectors1.1 Selection of the target sites of TβR-ⅡgeneBased on the mouse TβR-Ⅱgene (GenBank accession No. AF406755) sequence, three pairs of siRNAs to three different target sites were designed and synthesized, and named siRNA.1, siRNA.2 and siRNA.3.1.2 Selection of siRNAsThe three pairs of siRNAs were separately transfected to mouse fibroblast NIH3T3 cell line by Oligofectamine?. After 48h of transfection, cell total RNA and protein was extrcated for RT-PCR and Western Blot analysis to identify which pair of siRNA was the most effective one. Then this pair of siRNA was selected for the further experiments.1.3 Construction of MSCV-shRNAs retrovirus vectorCorrespondingly, shRNA to TβR-Ⅱgene, with the name of shRNA-T, was synthesized based on the selected siRNA sequence. Negative control shRNA, with the name of shRNA-N, was also synthesized. The two shRNAs were ligated into the linearized pEGFP/U6 vector by ApaⅠand EcoRⅠ. Then the whole U6-shRNA sequence was obtained by BamHⅠ/EcoRⅠdigestion and inserted into the MSCV-TβRⅡDN-IRES-GFP vector, which was also linearized by BamHⅠ/EcoRⅠdigestion. The reconstructed MSCV-shRNAs were named MSCV-shRNA-T and MSCV-shRNA-N respectively and were identified by DNA sequencing.2 Production of tumor-reactive TGF-β-insensitive CD8+ T cells2.1 Package and titration of MSCV-shRNAs retrovirusMSCV-shRNA and pVSV-G were cotransfected to pantropic GP293 retroviral packaging cells. After cotransfection at 37℃for 12h, the supernatant was replaced by fresh DMEM medium containing 10% FBS for additioanl incubation at 37℃, 5% CO2 for 48h. The supernatant, which contained MSCV-shRNA reconstructed retrovirus, was then collected for determination of virus tite or for cryopreservation at -80℃.2.2 Isolation, cultivation and identification of tumor-reactive CD8+ T cellsRenca tumors were established in BALB/c mouse. Splenic CD8+ T cells were isolated by using MagCellect* mouse CD8+ T cell isolation kit (R&D Systems) and cultured at 37℃, 5% CO2 in the presence of Renca lysates and irradiated autologous splenocytes in a medium containing RPMI-1640 with 10% FBS, rmIL-2 (50 U/ml), anti-CD3+ moloclonal antibody (30 ng/ml, R&D), HEPE (25 mM), L-glutamine (4 mM), and 2-ME (25 mM). The cell purity was determined by flow cytometry.2.3 Production of tumor-reactive TGF-β-insensitive CD8+ T cells by infection of MSCV-shRNAs retrovirusTumor-reactive CD8+ T cells were infected by MSCV-shRNAs retrovirus and incubated at 37℃, 5% CO2 for 48h. Three types of CD8+ T cells were established. The first type was tumor-reactive RNAi-induced TGF-β-insensitive CD8+ T cells (tumor-reactive CD8+ T cells infected with the MSCV-shRNA-T virus). The second type was tumor-reactive CD8+ T cells infected with the MSCV-shRNA-N virus. The third type was na?ve CD8+ T cells, which were freshly isolated from the spleen of na?ve donor animals without any treatment. The three types of CD8+ T cells were named M-T-CD8+T, M-N-CD8+T and N-CD8+T.2.4 The expression level of TβR-ⅡmRNA in each group of CD8+ T cells was determined by RT-PCR2.5 The expression level of TβR-Ⅱprotein in each group of CD8+ T cells was determined by Western Blot3 The killing effect of tumor-reactive RNAi-induced TGF-β-insensitive CD8+ T cells to mouse renal cancer Renca cell line in vitro and in vivo3.1 Western Blot analysis for the expression of SMAD-2 and P-SMAD in each group of CD8+ T cells3.2 Thymidine incorporation assay for the analysis to each group of CD8+ T cells proliferation in vitro3.3 Detection of each group of CD8+ T cells'killing effect on Renca cell line or an irrelevant mouse melanoma B16-F1 cell line in vitro by a standard 51Cr release assay3.4 Assessment of anti-tumor effect presented by tumor-reactive TGF-β- insensitive CD8+ T cells in vivoSubcutaneous Renca tumors were established in BALB/c mouse. Total of 30 BALB/c tumor-bearing mice were divided into 3 groups randomly and inoculated i.p. with each group of CD8+ T cells respectively. Tumor growth and mouse survival were monitored post-inoculation. Forty days later, all of the survival mice were sacrificed and serum levels of IL-2 and IFN-γwere determined by enzyme-linked immunoabsorbant assay (ELISA). Splenic CD8+ T cells were isolated and the percentage of GFP-positive ones was determined by flow cytometry.[Results]1 Selection of siRNAs and construction of MSCV-shRNAs retrovirus vectors Three pairs of siRNAs transfected to mouse fibroblast NIH3T3 cell line. After 48h of transfection, cell total RNA and protein was extrcated. The results of RT-PCR and Western-Blot indicated that the expression level of TβR-Ⅱin siRNA.1 transfected cells decreased significantly and that siRNA.1 was the most effective one. Correspondingly, shRNA to TβR-Ⅱgene, with the name of shRNA-T, was synthesized based on the selected siRNA sequence. Negative control shRNA, with the name of shRNA-N, was also synthesized. By use of gene engineering technology and DNA sequencing, MSCV retrovirus vectors containing shRNAs were successfully reconstructed and named MSCV-shRNA-T and MSCV-shRNA-N, respectively.2 Production of tumor-reactive TGF-β-insensitive CD8+ T cells2.1 Package and titration of MSCV-shRNAs retrovirusMSCV-shRNA and pVSV-G were cotransfected to pantropic GP293 retroviral packaging cells. Then the collected supernatant at different dilution, which contained MSCV-shRNA reconstructed retrovirus, infected NIH3T3 cells. The NIH3T3 cells with green fluorescence were observed by fluorescence microscope and the GFP-positive cell counts were determined by flow cytometry. The calculated virus tite was 7.15×1010VP/L.2.2 Production of tumor-reactive TGF-β-insensitive CD8+ T cellsSplenic tumor-reactive CD8+ T cells were isolated from BALB/c Renca tumor-bearing mouse by using MagCellect* mouse CD8+ T cell isolation kit (R&D Systems). The cell purity was 95.3% determined by flow cytometry. Tumor-reactive CD8+ T cells were infected with the MSCV-shRNAs retrovirus. The infection efficiency was determined by flow cytometry analysis. They were 93.1% and 91.6%, respectively, for the MSCV-shRNA-T and MSCV-shRNA-N retrovirus. MSCV-shRNA-T, MSCV-shRNA-N retrovirus infected tumor-reactive CD8+ T cells and na?ve CD8+ T cells freshly isolated from the spleen of na?ve donor animals without any treatment were established and named M-T-CD8+T, M-N-CD8+T and N-CD8+T respectively. 2.3 The expression level of TβR-Ⅱin each group of CD8+ T cellsRT-PCR and Western Blot indicated that TβR-Ⅱexpression in mRNA and protein level was decreased in the M-T-CD8+T. While in M-N-CD8+T and N-CD8+T, there was no significant decrease of TβR-Ⅱexpression. These results suggested that tumor-reactive TGF-β-insensitive CD8+ T cells were successfully established.3 The killing effect of tumor-reactive RNAi-induced TGF-β-insensitive CD8+ T cells to mouse renal cancer Renca cell line in vitro and in vivo3.1 Western Blot analysis for the expression of SMAD-2 and P-SMAD in each group of CD8+ T cellsSmad-2 and phosphorylated Smad-2 were detected by Western blot analysis after the three types of CD8+ T cells were treated with 10 ng/ml TGF-β1. The presence of Smad-2 was detected in all CD8+ T groups. But, phosphorylated Smad-2 was only detected in M-N-CD8+T and N-CD8+T in response to TGF-β1; absence of phosphorylated Smad-2 in M-T-CD8+T confirmed that TGF-βsignal transduction was successfully blocked by the MSCV-shRNA induced RNAi.3.2 Results of thymidine incorporation assayThe inhibitory rate of TGF-βon thymidine uptake was compared among the three types of CD8+ T cells after the addition of TGF-β1 for 72 h. TGF-β1 showed a dramatic antiproliferative effect on the established M-N-CD8+T and N-CD8+T, inhibiting uptake by a mean of 67.5% and 63.5% respectively. Whereas the mean inhibitory rate of thymidine uptake by M-T-CD8+T was 17%, the resistance to the antiproliferative effects of M-T-CD8+T was statistically significant when compared with the other groups (P<0.05). These results suggested that M-T-CD8+T were insensitive to the antiproliferative effect of TGF-β1.3.3 Detection of each group of CD8+ T cells'killing effect on Renca cell line in vitroThe ability of these CD8+ T cells to lyse Renca cells or irrelevant mouse melanoma B16-F1 cells was assayed in vitro using a standard 51Cr release assay. The results indicated that M-T-CD8+T showed the most potent Renca-specific CTL response (41% killing activity at an effector:target cell ratio of 100:1). M-N-CD8+T showed determinate Renca-specific CTL response (10% killing activity at an effector:target cell ratio of 100:1). N-CD8+T showed no significant CTL response. No apparent lysis was observed against irrelevant B16-F1 cells by these three types of CD8+ T cells. These results suggested that blocking TGF-βsignaling of tumor-reactive CD8+ T cells may improve the tumor-specific killing activity.3.4 Assessment of anti-tumor effect presented by tumor-reactive TGF-β- insensitive CD8+ T cells in vivoTo assess the antitumor effect of the M-T-CD8+T in vivo, Renca tumors were established in BALB/c mice. Compared with N-CD8+T group, adoptive i.p. transfer of M-T-CD8+T and M-N-CD8+T significantly suppressed the growth of the tumor (P<0.01,P<0.05,vs.control, respectively), with the M-T-CD8+T showing the more significant inhibitory effect. Complete tumor regression occurred in 20% of Renca-tumor-bearing mice that were treated with M-T-CD8+T. Forty days later, the survival rate of M-T-CD8+T, M-N-CD8+T and N-CD8+T treated mice was 80%, 30% and 20% respectively. Statistical analysis by using the Mantel-Haenszel log-rank test indicated a significant difference between the M-T-CD8+T and the other two control groups (P<0.01). These results demonstrated that the tumor-reactive TGF-β-insensitive CD8+ T cells were effective in improving the survival rate in mice bearing Renca tumors.Forty days after adoptive i.p. transfer of these three types of CD8+ T cells, all of the survival mice were sacrificed. Serum levels of IL-2 and IFN-γwere detected by enzyme-linked immunoabsorbant assay (ELISA). Compared with N-CD8+T treated group, the level of IL-2 and IFN-γin M-T-CD8+T and M-N-CD8+T treated mice increased significantly. A further increase in serum IL-2 and IFN-γwas observed in M-T-CD8+T group, suggesting the tumor-reactive TGF-β-insensitive CD8+ T cells promoted the host to product corresponding cytokines to kill the tumor cells robustly.Splenic CD8+ T cells were isolated and the percentage of GFP-positive ones was determined by flow cytometry. Adoptively transferred M-T-CD8+T cells were detected with a percentage of 2.0%, and 0.2% for M-N-CD8+T cells. The difference between the two groups was statistically significant, suggesting that these tumor-reactive TGF-β-insensitive CD8+ T cells were able to persist in recipient tumor-bearing hosts at least at the time of sacrifice, which occurred 40 days after the initial adoptive transfer.[Conclusions]1 We successfully constructed a retrovirus vector to deliver shRNA to mouse TβR-Ⅱgene.2 By use of the method to isolate CD8+ T cells from the tumor-bearing mice and make Renca cell lysate as antigens, we successfully induced the renal cancer reactive CD8+ T cells.3 MSCV retrovirus containing shRNA to TβR-Ⅱgene was used for the first time to infect renal cancer reactive CD8+ T cells. The TβR-Ⅱwas successfully knocked down so that the TGF-βsignaling was blocked.4 Tumor-reactive TGF-β-insensitive CD8+ T cells were insensitive to the antiproliferative effect of TGF-β1 and their tumor-specific killing activity was stronger.5 Tumor-reactive TGF-β-insensitive CD8+ T cells were able to persist for a period in recipient tumor-bearing hosts. These CD8+ T cells significantly suppressed tumor growth and increased survival rate of Renca tumor-bearing mice. Furthermore, complete tumor regression occurred in 2 treated mice. Serum levels of IL-2 and IFN-γin tumor-reactive TGF-β-insensitive CD8+ T cells treated mice were significantly improved. Taken together, by use of retrovirus induced RNAi to knock down TβR-Ⅱon the surface of tumor-reactive CD8+ T cells, the TGF-βsignaling was blocked, which decreased the inhibitory effect of TGF-βon the proliferation and differentiation of CD8+ T cells. Thus, the tumor-killing activity of CD8+ T cells were improved. It was demonstrated that reasonable use of RNAi technology to promote the tumor-killing activity of immune effctor cells could potentially be a new experimental approach for the immunogene therapy to renal cancer.