The Role Of Aberrant Frequency Of TNFR2~+Tregs,Related Cytokines And The Expression Of TNFR2 In The Progression Of Cervical Cancer

BackgroundIn 2012,the number of new cases of cervical cancer(CC)was 527,600,whereas the number of deaths due to this disease reached 265,700.CC has become the second most frequently diagnosed tumor and the third leading cause of cancer among women in developing countries.In China,an increasing prevalence of CC was found in young patients.Persistent infection of high-risk human papillomavirus(HPV)is the main cause of CC.Most patients who were infected by HPV could clear the virus through humoral or cellular immunity.Only a small group of theses patients will develop from CINI,CINII to CIN III,and finally become invasive cancer.Therefore,the immune status of the body and immune regulation play an important role in the occurrence and progression of cervical cancer.Cellular immunity mediated by T cells plays an important role in cancer immunity of cervical cancer.Previous studies have found that there is an abnormal increase in regulatory T cells(Treg)in cervical cancer patients,which inhibits the anti-tumor immune response of effector cell T cells(Teff).The immunosuppressive tumor microenvironment consisted mainly of Tregs is associated with tumor immune escape.In the past,it was clear that Treg cells could inhibit the functions of Teff cells,and whether Teff cells influence Treg cells remains unclear.Recently,Grinberg-Bleyer et al.found that Teff cells could promote the proliferation and differentiation of Treg cells through the binding of TNF-a expressed by Teff cells and the tumor necrosis factor receptor 2(TNFR2)expressed on the surface of Treg cells.Chen et al.showed that TNFR2 was predorminantly expressed by Treg rather than Teff.The binding of TNF-a to TNFR2 could thus promote the differentiation of Treg cells.TNFR2 combined with the simultaneous expression of CD4 and CD25,identifies the maximally suppressive subgroups of Tregs in both mice and human beings.The percentage of TNFR2+Treg increased in various tumors,for example,lung cancer,ovarian cancer and acute myeloid leukemia,which assisted immune escape and was related to poor prognosis.Studies show that the proportion of TNFR2+Treg subsets increased significantly in tumor infiltrating lymphocytes,and increased expression of TNFR2 may be an important feature of tumor-associated Treg cells.In a recent study,Govindaraj et al.found that the combined treatment of panobinostat and azacitidine could improve the prognosis of AML by lowering the proportion of TNFR2+Treg.Thus,further study of TNFR2+Treg and its possible role in the progression of cervical cancer could provide some new ideas in making future immunotherapeutic decisions.TNF-a is a pleiotropic cytokine and showed higher levels in various kinds of tumors,which plays multiple roles in regulating tumor immune effects.Most functions of TNF-a are activated through binding to TNFR1 and TNFR2.Different from TNFR1,TNFR2 lacks a death domain and is restricted expressed by several types of cells,such as immune cells,endothelial cells and neurons,mediating signaling pathways related to cell activation and proliferation.Soluble tumor necrosis factor receptors,s-TNFR2 and s-TNFR1,can be produced from shedding of the transmembrane tumor necrosis factor receptors by TNF-a converting enzymes or non-classical splicing transcripts.Both s-TNFR2 and s-TNFR1 can modulate the biological function of TNF-a through a competitive binding or a reversible binding to TNF-?,which plays an important role in various inflammatory diseases.s-TNFR2 and s-TNFR1 are found significantly elevated in various kinds of tumors,such as colorectal cancer,non-Hodgkin's lymphoma,which are associated with poor prognosis.Whether s-TNFR2 and s-TNFR1 participate in the occurrence and development of CC remains unclear.Recent studies showed increased expression of TNFR2 in several tumors,for example,multiple myeloma,Hodgkin's lymphoma,colon cancer,renal cell carcinoma,which was related to the poor prognosis of the disease.TNFR2 could promote the proliferation of tumor cells under the stimulation of several inflammatory factors within the tumor microenvironment,such as IL-6 and TNF-a,through NF-kB signaling pathways.Pallavi et al.reported that the gene polymorphism of TNFR2 is associated with the risk of cervical cancer.However,the expression of TNFR2 protein in cervical cancer and their effects on the biological behavior of cervical cancer cells remain uncovered.It was showed that on the one hand,TNFR2 could accelerate tumor immune escape through promoting the proliferation and activation of Tregs,on the other hand,TNFR2 could directly promote tumor progression as an oncogenic protein highly expressed by tumor cells.The restricted expression of TNFR2 making it a promising target for future therapies.Targeting TNFR2 could benefit both in controlling tumor immune escape and inhibiting the proliferation and metastasis of tumors.In the present study,we examined the level of TNFR2+Tregs in both peripheral blood(PB)and tumor infiltrating lymphocytes(TILs),relevant cytokines in patients with cervical intraepithelial neoplasia(CIN)III and different stages of CC and the expression level of TNFR2 within CC tissues.We also analyze the relationship between TNFR2+Tregs,s-TNFR2,TNFR2 and clinicopathological factors of CC.This study aimed to explain the role of TNFR2+Tregs,TNFR2 and related cytokines in the development of CC and to provide information for the manipulation of Treg cells in future immunotherapeutics.Part 1 Aberrant frequency of TNFR2+Tregs in patients with CIN and cervical cancerObjective1.To detect the proportion of TNFR2+Treg subsets in peripheral blood and tumor-infiltrating lymphocytes in patients with cervical cancer.To analyze the relationship between TNFR2+Treg subgroups and clinicopathological parameters of cervical cancer and to explore the role of TNFR2+Treg subpopulations in the occurrence and progression of cervical cancer.2.To detect the expression of TNFR2 on Treg and Teff cells,and the proportion of TNF-?+Thl7 cells in cervical cancer patients.To investigate the effect of TNF-a/TNFR2 pathway on the differentiation of Treg cells in vitro and explore the possible mechanisms affecting differentiation of TNFR2+Treg subsets.3.To investigate the effect of TNF-a on the immunosuppressive function of Treg cells by a co-culture system of Treg cells and Teff cells in vitro.MethodsPeripheral blood mononuclear cells(PBMCs)were collected from newly diagnosed cervical cancer patients,cervical precancerous lesions(CIN)patients and healthy volunteers from Qilu Hospital of Shandong University.Cervical cancer tissue samples from patients with newly diagnosed cervical cancer were collected to isolate tumor infiltrating lymphocytes.The percentage of CD4+CD25+TNFR2+ Treg subsets in peripheral blood and tumor-infiltrating mononuclear cells within CD4+T cells was measured by flow cytometry.The clinical stage,surgical pathology data of all patients were analyzed,and the correlation between each clinicopathological index and the ratio of TNFR2+ Treg cell subsets was analyzed.PBMCs of cervical cancer patients were isolated and cultured in vitro.IL-2 was added to maintain the growth,and PBMCs were stimulated by TNF-a and TNFR2 monoclonal antibodies respectively.After 3 days of culture,flow cytometry was used to detect the proportion of CD4+CD25+FOXP3+Treg cells.Magnetic beads were used to sort Treg(CD4+CD25+)and Teff(CD4+CD25-)cells from peripheral blood of patients with cervical cancer.Tef cells were labeled with CFSE and cultured with Treg in vitro for 5 days.Flow cytometry was used to detect the proliferation of Teff cells to investigate the effect of TNF-a on the immunosuppressive function of Treg cells.Results1.The proportions of CD4+CD25+Treg cells and TNFR2+Treg cells were higher in cervical cancer patients and CIN patients compared with healthy volunteers(P<0.001).2.In patients with cervical cancer,the proportion of TNFR2+Treg subsets in locally infiltrating lymphocytes was significantly higher than that in peripheral blood(P<0.001).3.The proportion of TNFR2+Treg subsets in patients with stage II CC showed a decreasing trend compared with those with stage I CC(P<0.05).There were no significant correlations between the percentage of TNFR2+Tregs and tumor size,differentiation degree and lymph node metastasis.4.As the main subpopulations of Teff that express TNF-?,the proportion of TNF-a+Th17 cells in the peripheral blood of cervical cancer group was significantly higher compared with control group.TNF-a could promote the differentiation of Treg cells in the in vitro culture of PBMCs.Blocking of TNFR2 can reduce the effect.5.Constructing a co-culture system of Treg cells and Teff cells in vitro,TNF-a had no significant effect on the immunosuppressive function of Treg cells.Conclusions1.The aberrant frequency of TNFR2+Treg subsets in patients with CIN and CC suggests that TNFR2+Treg play a role in tne oncogenesis and development of CIN and cervical cancer.2.TNF-a can promote the differentiation of Treg cells through the TNF-a/TNFR2 pathway.Elevated TNF-a+Th17 subsets in the peripheral blood of patients with cervical cancer may play a role in promoting the differentiation of Treg subsets through the interaction between TNF-? expressed by Th17 and TNFR2 on Treg.3.TNF-a could not enhance the immunesuppressive function of Treg.Part 2 The plasma levels of soluble TNFR in patients with CIN and cervical cancer and its significanceObjective1.To detect the plasma levels of s-TNFR2,s-TNFRl in patients with CIN,cervical cancer and healthy controls.To analyze the relationship between s-TNFR2,s-TNFRl and clinicopathological characteristics of cervical cancer patients and to explore the relationship between plasma levels of s-TNFR2 and s-TNFR1 and the oncogenesis of cervical cancer.2.To analyze the correlation between the level of s-TNFR2 and the proportion of TNFR2+Treg in cervical cancer patients.To explore the TNFR2,TNF-a,TNFR1 and FOXP3 mRNA expression in patients with CC?CIN and healthy controls,and to analyze the relationship between clinicopathological characteristics and expression level.MethodsPeripheral blood samples from 72 patients with cervical cancer,30 patients with CINIII and 30 healthy women were collected and serum was separated.Serum levels of s-TNFR2 and s-TNFR1 were detected by enzyme-linked immunosorbent assay(ELISA).RT-PCR was performed to detect the expression of TNFR2,TNF-a,TNFR1 and FOXP3.The relationship between serum levels of cytokines and clinicopathological characteristics of cervical cancer was analyzed.Results1.Compared with healthy controls,the plasma concentrations of s-TNFR2 and s-TNFR1 in patients with CIN and cervical cancer showed an increasing trend.(P<0.05)2.The expression levels of plasma a-TNFR2 and s-TNFR1 in the cervical cancer group had no significant correlation with clinicopathological parameters such as the stage of cervical cancer,tumor size,differentiation and metastasis of lymph nodes.3.The plasma level of s-TNFR2 was not related to the percentage of TNFR2+Treg.4.The expression levels of TNFR2,TNF-a and FOXP3 in mRNA in patients with CIN and cervical cancer were higher than those in healthy controls,while the level of TNFR1 mRNA was not statistically different among three groups.5.The mRNA expression level of TNFR2 was correlated with clinical stages,lymph node metastasis and involvement of lymphatic vessel space.The mRNA expression level of TNF-? is related to the degree of tumor differentiation.The mRNA expression level of FOXP3 is related to tumor stages.Conclusions1.The levels of plasma inflammatory cytokines,s-TNFR2 and s-TNFR1,were significantly increased in the cervical cancer group compared with the control group,suggesting that they were related to the occurrence of cervical cancer.2.There was no significant correlation between plasma levels of s-TNFR2 and s-TNFR1 and clinicopathological data in patients with cervical cancer,suggesting that s-TNFR2 and s-TNFR1 were not directly related to the progression of cervical cancer.3.The s-TNFR2 in plasma was mainly shed by peripheral TNFR2+Treg.Levels of s-TNFR2 in patients with cervical cancer was not significantly related to the percentage of TNFR2+Treg,which indicated that plasma s-TNFR2 levels were also regulated by other factors.4.The expressions of TNFR2,TNF-? and FOXP3 were increased at the transcriptional level and were related to the clinical characteristics of CC,which suggested they played a role in the development of CC by regulating TNFR2+Treg and s-TNFR2.Part 3 The expression of TNFR2 in cervical cancer tissues and its effect on biological behaviors of cervical cancer cellsObjective1.To detect the expression of TNFR2 in cervical cancer tissues and normal cervical tissues.To analyze the correlation between the expression level of TNFR2 in cervical cancer and the clinicopathological parameters of cervical cancer patients,and to explore the correlation between TNFR2 and the occurrence and progression of cervical cancer.2.To detect the proliferation,migration and invasion of cervical cancer cell lines after the interference of TNFR2 expression.To examine the expression levels of I?B?,p-I?B?,NF-?B,and p-NF-?B in cervical cancer cell lines after the interference of TNFR2 expression.To investigate the effect of TNFR2 on the biological behavior of cervical cancer cells and the underling mechanism.MethodsTotally 68 cases of cervical cancer tissues and 20 cases of normal cervical tissues were collected during surgical resections.The expression of TNFR2 protein was detected by immunohistochemistry(IHC)method.The relationship between the clinicopathological characteristics of the patients and the expression of TNFR2 was analyzed by Pearson chi-square test or Fisher's test.After siRNA was used to inhibit the expression of TNFR2,the proliferation of cervical cancer cell lines,HeLa and CaSki,was detected by CCK 8 and the migration and invasion ability was detected by Transwell chamber.Western Blot was used to examine the protein level of I?B?,p-I?B?,NF-?B,and p-NF-?B in cervical cancer cells.Results1.IHC results showed that TNFR2 was highly expressed in cervical cancer tissues and barely detectable in normal cervical tissues.TNFR2 was mainly located in the cytoplasm of cervical cancer cells.In 68 cases with cervical cancer,high expression of TNFR2 accounted for 69.1%(47/68),and low expression of TNFR2 accounted for 30.9%(21/68).2.The expression of TNFR2 was not significantly associated with the clinicopathological parameters such as stage,size,differentiation and metastasis of lymph nodes in patients with cervical cancer.3.After the expression of TNFR2 was interfered,the proliferations of HeLa and CaSki in vitro were significantly decreased compared with the control group under TNF-?(50ng/mL)stimulation(P<0.05).Transwell experiments showed that the migrations and invasions of HeLa and CaSki were significantly inhibited compared with the control group(P<0.01).4.Western Blot showed that under the stimulation of TNF-a,compared with the negative control group,there was no significant difference in levels of NF-?B and I?B-? and there were lower levels of p-NF-?B and p-?B-a after the interference of TNFR2 expression.(P<0.05)Conclusions1.TNFR2 was highly expressed.in cervical cancer tissues compared with normal cervical tissues,suggesting that TNFR2 is involved in the oncogenesis of cervical cancer.2.No significant difference between the expression level of TNFR2 and clinicopathological parameters of cervical cancer was found,which may be caused by limited case numbers and failures in including patients with advanced cervical cancer.3.In vitro,TNFR2 could promote the proliferation,migration,and invasion of cervical cancer cells under TNF-? stimulation through the activation of NF-?B signal pathway.