The Negative Regulation Of Regulatory T Cell To Tumor Immune Microenvironment In Renal Cell Carcinoma And Its Underlying Mechanism

Partâ… The prognostic value of peritumoral regulatory T cell and its correlation with intratumoral cyclooxygenase-2 expression in renal cell carcinomaObjective This study aims to investigate the prognostic value of various tumorinfiltrating lymphocytes(TILs) in renal cell carcinoma(RCC).Furthermore,we intend to know whether they are also associated with the clinical parameters in RCC.In addition,by elucidating the prognostic value of regulatory T cell(Treg) and its correlation with cyclooxygenase-2(COX-2) expression,we try to know the possible mechanism of aberrant gathering of Treg in RCC.Methods In this study we identified 125 patients with RCC treated with radical nephrectomy or nephron-sparing surgery for unilateral,sporadic,clear cell RCC at Zhong Shan Hospital of Fudan University from Jan 1999 to Dec 2005.Then CD4⁺, CD8⁺,Granzyme B⁺,FOXP3⁺ TILs and tumour COX-2 expression were assessed by immunohistochemistry in tissue microarrays.The prognostic effects of low and high expression were evaluated and the expression of various immunohistochemical parameters were compared with the clinicopathological variables.In addition,Treg and its correlation with COX-2 expression was also analysed.Results The results showed that(1) immune cell infiltration varied substantially among different patients.Lymphocytes infiltrated RCC in a diffuse manner or in lymphoid aggregates,with more abundant cells in peritumoral areas.(2) On univariate analysis,age and sex had no prognostic significance for overall survival nor disease-free survival.CD4⁺,CD8⁺,Granzyme B⁺ T cells were associated with neither overall survival nor disease-free survival,while peritumoral Treg is an prognostic factor for both overall survival and disease-free survival(P＜0.001,P＜0.001).Similarly, high intratumoral COX-2 expression was also considered to be prognostic for both reduced overall survival and disease-free survival(P=0.021,P=0.027).Nevertheless, both peritumoral COX-2 and intratumoral Treg had no prognostic value in RCC. Tumor TNM stage,tumor nuclear grade and tumor size could also be prognostic in RCC.(3) Using multivariate analysis,increased peritumoral Treg,higher TNM stage (â…¢,â…£),higher nuclear grade(3,4) and larger tumour size(≥7cm) were independent predictors for significant shorter overall survival and disease-free survival.In addition, peritumoral Treg were positively associated with TNM stage and tumor size(P=0.001, P=0.002),granzyme B⁺ TILs were positively associated with nuclear grade(P=0.024), while intratumoral COX-2 expression were positively associated with TNM stage and nuclear grade(P=0.018,P=0.013).Peritumoral Treg were positively correlated with intratumoral COX-2 expression(P＜0.001).Conclusions These results suggested that increased peritumoral Treg,higher TNM stage(â…¢,â…£),higher nuclear grade(3,4) and larger tumour size(≥7cm) were independent predictors for significantly shorter overall survival and disease-free survival.In the present study peritumoral Treg was positively correlated with intratumoral COX-2 expression.In addition,a previous report in lung cell carcinoma showed the important role of COX-2 derived prostaglandins E₂(PGE₂) in the transformation of Treg.Similarly,the overexpression of COX-2 in RCC could be the underlying reason for the aberrant gathering of Treg.Therefore,we should further elucidate the possible mechanism of the aberrant gathering of Treg in RCC.Partâ…¡The origin of regulatory T cell in renal cell carcinoma and its possible undelying mechanism2.1 Construction of eukaryotic expression vector pcDNA3.1-hCOX-2Objective To construct eukaryotic expression vector of pcDNA3.1-hCOX-2.Methods The hCOX-2 cDNA fragment was extracted from plasmid pSG5-COX-2,amplified by PCR,and inserted into pcDNA3.1 vector before the double digestion with restriction enzymes EcoRI and BamHI.Then the vector was identified by the double digestion with restriction enzymes and was sequenced by the Sanger-dideoxy-mediated chain termination.Results The recombinant eukaryotic expression vector for COX-2 was digested with EcoRI and BamHI,and the electrophoresis of the digested products showed two fragments:1800 bp fragmant and 5.4 kb fragment.The sequence of 1800 bp fragment was identical to COX-2 sequence published in GeneBank.Conclusions The recombinant plasmid pcDNA3.1-hCOX-2 was constructed successfully and can be applicated into subsequent transfection experiment. 2.2 The increased COX-2 expression and PGE₂ production in the transfected RCC cell line after pcDNA3.1-hCOX-2 transfectionObjective To detect the expression of COX-2 gene and protein in RCC cell lines and pick out RCC cell line with higher expression of COX-2 for the following transfection experiment.Next we transfect pcDNA3.1-hCOX-2 plasmid vector into the targeted RCC cell line,then we analyze its effect to the expression of COX-2 gene and protein,especially we tend to know its effect to the production of PGE₂ in the RCC cell line supematants.In addition,we add different doses of NS-398 into the transfection group to know their effect to the production PGE₂ in the supernatants.Methods(1) Two RCC cell lines 786-0 and OS-RC-2,were cultured in vitro. The expressions of COX-2 mRNA were assessed by RT-PCR,and its protein expressions were determined by Western-blot.The results were compared with each other to determine which cell line can be used in the next part of experiments.(2) To transfect pcDNA3.1-hCOX-2 plasmid vector into OS-RC-2 using lipofectamine 2000,COX-2 mRNA were detected 72h after transfection by RT-PCR and COX-2 protein were analysed by Western-blot 72h after transfection.(3) The different levels of PGE₂ in the transfected OS-RC-2 cell line supernatants and control groups were analysed by ELISA.In addition,we added 12.5μM,25μM,50μM,100μM NS-398 among the supernatants respectively and detected the PGE₂ levels by ELISA.Results(1) The two RCC cell lines had different COX-2 mRNA and protein expression.RT-PCR:The COX-2/beta-actin ratio was used to represent the expression of COX-2 mRNA.The ratio of 786-0 and OS-RC-2 cell lines were 0.2756±0.01318 and 0.4667±0.02159 respectively.It represented that the expression of COX-2 mRNA of 786-0 was about 59.1%compared with that of OS-RC-2 cell lines.The results between 786-0 and OS-RC-2 cell lines had significant difference(p＜0.05). Western-blot:The COX-2/GAPDH ratio was used to represent the expression of COX-2 protein.The ratio of 786-0 and OS-RC-2 cell lines were 0.2333±0.02183 and 0.4686±0.04294 respectively.It represented that the expression of COX-2 protein of 786-0 was about 50%compared with that of OS-RC-2 cell lines.The results between 786-0 and OS-RC-2 cell lines had significant difference(p＜0.05).Next we chose OS-RC-2 as the targeted cell line for the next transfection experiment.(2) The expression of COX-2 mRNA and protein in the transfected OS-RC-2 cell line have increased significantly after pcDNA3.1-hCOX-2 plasmid vector transfection.We divided into three groups:pcDNA3.1-hCOX-2,pcDNA3.1 and control group.Results of RT-PCR:COX-2 mRNA was detected 72h after transfection.The COX-2mRNA in the above three groups were 0.7922±0.07139,0.4325±0.02045,0.4739±0.02377 respectively.Compared with pcDNA3.1 and control groups,the transfection group had higher COX-2mRNA expression(p＜0.05),while the COX-2 mRNA between pcDNA3.1 and control group had no significant difference(p＞0.05).Results of Western-blot:COX-2 protein was detected 72h after transfection.The COX-2 protein in the above three groups were 0.8593±0.1332,0.4321±0.03362,0.4533±0.05883 respectively.Compared with pcDNA3.1 and control groups,the transfection group had higher COX-2 protein expression(p＜0.05),while the COX-2 protein between pcDNA3.1 and control group had no significant difference(p＞0.05).(3)The result of ELISA experiment indicated that PGE₂ levels varied significantly among pcDNA3.1-hCOX-2,pcDNA3.1 and control group.The PGE₂ levels in the above three groups were 6.330±1.181,0.2817±0.03181,0.3030±0.03246 respectively. Compared with pcDNA3.1 and control groups,the transfection group had higher PGE₂ production(p＜0.05),while PGE₂ production between pcDNA3.1 and control group had no significant difference(p＞0.05).When applicating NS-398 at 12.5μM,25μM,50μM,100μM respectively in pcDNA3.1-hCOX-2 transfection group,the PGE₂ levels were 2.906±0.5892,0.6484±0.09880,0.4189±0.06513,0.2221±0.04094 respectively.Thus,the PGE₂ production between these groups and pcDNA3.1-hCOX-2 transfection group had significant difference(p＜0.05).Conclusions(1) The two RCC cell lines had COX-2 mRNA and protein expression.Since OS-RC-2 cell line had higher COX-2 expression,we chose OS-RC-2 as target cell line for the following transfection experiment.(2) The constructed pcDNA3.1-hCOX-2 could be successfully transfected into OS-RC-2 cell line using lipofectamine 2000,and subsequently increased the COX-2 expression.(3) The production of PGE₂ in the transfected OS-RC-2 cell line increased significantly. The application of NS-398 could reduce PGE₂ production at the supernatants in a dose-dependent manner.2.3 The increased conversion from CD4⁺FOXP3^- T cell to CD4⁺FOXP3⁺ Treg in the supernatants of pcDNA3.1-hCOX-2 transfection groupObjective To investigate whether the supernatants from cultured transfected OS-RC-2 cell line could convert peripheral CD4⁺FOXP3^- T cell into CD4⁺FOXP3⁺ Treg. Methods We obtained peripheral blood mononuclear cells by density gradient centrifugation method.Subsequently we isolated CD4⁺CD25^- T cell and CD4⁺ CD25⁺ Treg by MACS and analysed the purity of isolated cells by RT-PCR and FACS respectively.Under the stimulation of anti-CD3/CD28 antibody and APC cells, isolated CD4⁺FOXP3^- T cells were cocultured with transfected OS-RC-2 supernatants and different control supernatants respectively,96 hours later,the proportion of CD4⁺FOXP3⁺ Treg in each group were detected by FACS.Results(1) MACS could obtain CD4⁺CD25- T cell and CD4⁺CD25⁺ Treg steadily and the purity of two groups were over 90%respectively.The latter characteristically expressed FOXP3 gene and protein.(2) The transfected OS-RC-2 cell line supernatants could obviously induce the production of CD4⁺FOXP3⁺Treg, the proportion of CD4⁺FOXP3⁺Treg reached 30.00±2.618%after 96 hours coculture, while NS-398 could markedly reduce the above effect,the proportion of CD4⁺ FOXP3⁺ Tregs was only 7.990±1.227%in the transfection+100μMNS-398 group.Conclusions(1) MACS could obtain CD4⁺CD25^-T cell and CD4⁺CD25⁺ Treg steadily.Since CD4⁺CD25⁺ Treg specifically express FOXP3 gene and protein,we regard CD4⁺CD25⁺Treg as CD4⁺FOXP3⁺Treg,while CD4⁺CD25^- T cell do not express FOXP3 gene and protein,we regard CD4⁺CD25^-T cell as CD4⁺FOXP3^- T cell in the following experiment.(2) The transfected RCC cell line supernatants could induce the transformation from CD4⁺FOXP3^- T cells to CD4⁺FOXP3⁺ Treg,while NS-398 could reduce the proportion of induced CD4⁺FOXP3⁺Treg.Therefore, COX-2 inhibitors may induce the local anti-tumor effect and in turn contribute to eradicating RCC.