The Influence Of Immunosuppressive Factors On Cervical Carcinoma After Radiation And Its Regulation

Objective:(1)By testing the expression of PD-1,PD-L1 between cervical cancer and benign disease patients and their matched blood sample,combined with clinical data to investigate the correlation of the expression level and the prognosis.(2)To understand the changes of PD-1,PD-L1 and EBI3 in Wistar mice bearing Hela Hela cell transplantation tumor model after irradiation.(3)To understand the changes of T,Treg cell proliferation,apoptosis,cell cycle,and PD-1/PD-L1 in Hela cell line through the expression of EBI3 or inhibiting EBI3 expression.Methods:(1)70 cases of cervical squamous cell carcinoma and cervical benign lesion were collected,and 24 cases were matched with peripheral blood squamous cell carcinoma,30 cases were benign lesions.Immunohistochemistry method was used to detect the expression of PD-L1 in the tissues.Flow cytometry was used to detect the proportion of CD4+PD-1+ and CD8+PD-1+ subsets in peripheral blood.The above information and the patient’s clinical pathological characteristics,survival data to do statistical analysis is employed to analyze the information.(2)Hela cells were inoculated into the back of Wistar mice,and the subcutaneous transplanted tumor model was established.The 43 rats were divided into radiotherapy group(n=31)and control group(n = 12).The radiotherapy group was given 6MeV electron irradiation.The tail vein blood was collected at 1 week and 1 months after the radiotherapy in the radiotherapy group and the control group,CD4 and CD8 were measured by flow cytometry after Ficoll separation of peripheral blood.The expression of EBI3,PD-1 and PD-L1 in tumor tissue was detected by immunohistochemistry.(3)To establish EBI3 overexpression and EBI3 inhibition of Hela cell lines by lentivirus plasmid transfection first.Tregs and T cells were collected from the spleen cells of Wistar mouse,which were co cultured for 72 hours with the cell lines.The Western Blot was used to detect the expression of EBI3,PD-1 and PD-L1 in EBI3 overexpression group,EBI3 inhibition group,negative control group and blank vector group.MTT method was used to test the proliferation of Tregs by the light density.Flow cytometry was used to detect the apoptosis rate of Tregs,CD4+T and CD8+T cells,and the ratio of CD4+T and CD8+T cells counting for CD3+T cells.Results:(1)There was no difference among age,marriage age,times of pregnancy,age of menarche,menopause,ethnic cultural level and occupation,except for family history of cancer.The expression of PD-1 in peripheral blood positively correlated with the expression of PD-L1 in tissue samples of cervical squamous cell carcinoma patients(R2=0.734,P=0.000;R2=0.66,P=0.000),while there were less correlation in cervical benign patients(R2=0.138,P=0.043;R2=0.174,P=0.022).There was significantly difference between cervical cancer group and cervical benign disease group of CD8+PD-1+T cells in peripheral blood and PD-L1+ cancer cells in tissue(P=0.000,P=0.002).The number of CD8+PD-1+ cells correlated with the status of menopause(chi square =7.152,P=0.012).There was no relationship among the tumor size,differentiation degree,depth of invasion,and the degree of PD-1 expression in peripheral blood cervical cancer group and cervical benign disease group.With the increase of tumor stage,the percentage of CD4+PD-1+ T cells and CD8+PD-1+ T cells increased correspondingly.With the increase of tumor diameter and stage,the expression of PD-L1 in tumor tissue also increased.The patients with low expression of CD4+PD-1+ T cells in peripheral blood and low expression of PD-L1 in tissue had longer survival time.Multivariate analysis showed that the PFS time was affected by the expression of PD-L1(B=1.844,P=0.019).(2)After radiotherapy,tumor diameter was smaller than before,but 1 month after radiotherapy,some tumors continued to grow.Compared with before and 1 week after radiotherapy,the percentages of CD4+T cells and CD8+T cells were significantly decreased in the radiotherapy group at 1 month after radiotherapy.Furthermore,down-regulation of EBI3 and up-regulation PD-1 and PD-L1 were observed in cervical cancer tissues at 1 month after radiotherapy.(3)In comparison to the blank and NC groups,increased expression of EBI3 and decreased expressions of PD-1 and PD-L1 were found in the EBI3 mimics group.However,the EBI3 inhibitors group had lower expression of EBI3 and higher expressions of PD-1 and PD-L1 than those in the blank and NC groups.The EBI3 mimics group showed an increase of the optical density(OD)value(0.43±0.05),while a decrease of the OD value(0.31±0.02)was found in the EBI3 inhibitors group.Moreover,compared with the blank and NC groups,the apoptosis rates of Treg/CD4+T/CD8+T cells were decreased in the EBI3 mimics group,but the EBI3 inhibitors group exhibited an increase of apoptosis rate.Conclusions:(1)The cervical squamous cancer patients had more CD4+PD-1+ T cells in peripheral blood and PD-L1+ cells in tissues,which correlated to the grading and prognosis of the tumor.(2)Radiotherapy could reduce the size of the xenograft tumor of Hela cells in Wistar mice,suggesting that Hela cells were sensitive to radiotherapy.The proportion of CD8 and CD4 T cells decreased significantly one month after radiotherapy,suggesting that T cells were inhibited after radiotherapy.The expression of PD-1 and PD-L1 increased one month after radiotherapy,suggesting that radiotherapy might cause immunosuppression in the tumor microenvironment.The expression of EBI3 decreased one month after radiotherapy,suggesting that radiotherapy could hava an influence on the expression of EBI3.(3)Over expressed EBI3 could decrease the expression of PD-1 and PD-L1,inhibit apoptosis of Tregs and CD4+/CD8+ T cells,and vice versa,which suggested that EBI3 negatively regulated PD-1 and PD-L1 expression,and negatively regulated the apoptosis of Treg and T cells.Moreover the function of EBI3 didn’t include the regulation of cell cycle of Treg or the proportion of CD4/CD8 cells.(4)Overexpression of EBI3 could reduce the apoptosis of Treg/CD4+T/CD8+T cells and prevent radiation-induced immunosuppression of cervical cancer He La cells by inhibiting the activation of PD-1/PD-L1 signaling pathway.