Expression Of B7-h1 In Esophageal Carcinoma And Its Impact On T Cell Function

The evasion of tumor immunity is a major mechanism involving in carcinogenesis of esophageal carcinoma.Co-stimulatory molecules participate in regulating immune response.B7-H1 is a new identified member of the B7 family molecules.PD-1 is a receptor of B7-H1 and is shown to mediate the inhibition of activated T cell response. However,there is ample evidence indicating that B7-H1 still has an unidentified non-PD -1 receptor.Upon ligation to its receptors on T cells B7-H1 regulates proliferation and differentiation of T cells.Expression of B7-H1 mRNA is found in a broad range of human normal tissues.But,expression of human B7-H1 protein is limited to the macrophage-derived cells.Interestingly,B7-H1 surface expression can also be found on many tumor tissues.In vitro experiments,it is indicated that many tumor cell lines also express B7-H1 and/or up-regulate B7-H1 surface expression upon exposure to IFN-γ. There is the hypothesis that tumors escape from the host immune system by negative attenuation of tumor-specific T-cell responses via the B7-H1/PD-1 pathway.However,expression of B7-H1 in esophageal carcinoma has not been well documented.Furthermore,the effect of B7-H1 on esophageal carcinoma specific T cells has not been studied so far.In this study,we investigated expression of B7-H1 in human esophageal cancer cell line and esophageal carcinoma tissues.Moreover,the association between B7-H1 protein expression and the patients' clinicophathological variables were analyzed.We also further explored the impact of B7-H1 on the function of esophageal carcinoma-specific T cells.Methods1.Cell culture Human esophageal cancer cell line EC9706 was routinely cultured in RPMI-1640 medium supplemented with 10%heat-inactivated FBS,in a 37℃,5%CO₂ incubator.2.Analysis of B7-H1 expression on esophageal cancer cell line EC9706The expressions of B7-H1 mRNA and B7-H1 protein were detected by RT-PCR, immunohistochemistry and flow cytometric analysis,respectively.3.Analysis of B7-H1 expression in esophageal tissuesRT-PCR,immunohistochemistry and Western blot were used to detect the expression of B7-H1 mRNA and protein in 54 cases of esophageal carcinoma tissues and 8 cases of adjacent normal esophageal tissues.Furthermore,we examined the association between B7-H1 protein expression and the patients' clinicopathological variables.Semi-quantitative results of the expression of B7-H1 mRNA and protein in 54 cases of esophageal carcinoma tissues were analyzed by Syngene and TotolLab 2.0 softwares, respectively.We further analyzed correlation between B7-H1 mRNA expression and B7-H1 protein expression in 54 cases of esophageal carcinoma tissues.4.Co-culture experiments of T cellsT cells were isolated from esophageal carcinoma tissues by discontinuous Percoll density gradient centrifugation.Then obtained T cells were cultured in 24-well plates which were pre-coated with soluble B7-H1 Ig and anti-CD3 mAb for a period of time.In the following,immune function of T cells was investigated.Control was designed by precoated only with anti-CD3 mAb.5.Cytokine assaysForty-eight hours later,cytokines in culture supernatants were collected,and the concentration of IL-10 or IFN-γwas assayed using commercial available ELISA kits.6.Statistical analysisStatistical analysis was performed using the SPSS 13.0 software.The association between B7-H1 expression and the clinicopathological variables was analyzed using the x² test.Mean values were compared using student's t test.The correlation between B7-H1 mRNA expression and B7-H1 protein expression was analyzed using Spearman's rank correlation.The significance level was set at 0.05,and all P values were two-sided. Results1.B7-H1 expression on esophageal cancer cell lineThe expression of B7-H1 mRNA was demonstrated in esophageal cancer cell line EC9706.And B7-H1 protein was located in the cell member,cytoplasm,or both.The percentage of B7-H1-positive cells on esophageal cancer cell line EC9706 was 28.88±3.22%(mean±SD).2.B7-H1 abnormal expression in esophageal carcinoma tissuesResults of RT-PCR,Western blot and immunohistochemistry showed that esophageal carcinoma tissues were positive for B7-H1 mRNA expression and B7-H1 protein expression.B7-H1 protein was expressed in a focal or scattered pattern.We found that 26 of the 54(48.15%) esophageal carcinoma tissues were positive for B7-H1 protein expression.However,normal esophageal tissues were negative for B7-H1 mRNA expression and B7-H1 protein expression.Furthermore,we found that B7-H1 mRNA expression was highly correlated with B7-H1 protein expression in 54 cases of esophageal carcinoma tissues(r=0.801,p＜0.05).3.Association between B7-H1 protein expression and the clinicophathological variables in esophageal carcinomaWe analyzed the association between the percentage of tumor-associated B7-H1 protein expression and clinicophathological variables of the 54 esophageal carcinoma cancers.No association was observed between B7-H1 expression and sex,age,tumor size, location,or differentiation(p＜0.05).Conversely,the expression of B7-H1 was significantly associated with depth of invasion,lymph node metastasis,or distal metastasis or pT classification(p＞0.05).4.Results of ELISAIn vitro experiment,we detected that immortalized B7-H1-Ig can promote IL-10 secretion and inhibit IFN-γsecretion in esophageal cancer-specific T cells in the presence of anti-CD3 mAb comparing with in the presence of only anti-CD3 mAb as a control.The difference reached statistical significance.Conclusions 1.B7-H1 high expression on esophageal carcinoma cells probably promotes carcinogenesis of esophageal carcinoma through down-regulating the specific anti-tumor immune responses.2.B7-H1 may be a novel indicator for forecast of metastatic potential or judgement of prognosis of esophageal carcinoma,and a potential immunotherapeutic target in future.3.B7-H1 inhibits anti-tumor immune function of esophageal cancer-specific T cells through up-regulating IL-10 secretion and down-regulating IFN-γsecretion.