The Mechanisms Study Of Overexpression Of Indoleamine 2,3-dioxygenase In Cervix Cancer And The Tumor Immune Evasion

Objective:With approximately 450,000 newly diagnosed cases each year and 50% mortality rate, cervix cancer is the second most common cause of cancer-related deaths in women worldwild. Cervical intraepithelial neoplasia(CIN) and cervical cancer are almost always assosiated with human papillomavirus(HPV). The cytotoxicity T lymphocytes , (CTL) play the important role in HPV association tumor immunosurveillance. Only in recent years, however, a tryptophan-degrading enzyme with well know suppressing T cell activity was show to be produced by cancer cells. It would be one of mechanisms of tumor immune escape. Up to the present, the characteristics of IDO expression and its functional significance in cervix cancer progression remains to be clarified. In the current study, we investigated the characteristics of the IDO expression on the cervix cancer and explored the mechanism by three parts. The first part included two clinical studys focused on the expression of IDO in cervix cancer tissue and lymph node metastatases tissue from patients. we show the correlation between the expression of IDO and clinical stages; differentiation degree and high risk human papillomavirus(HR-HPV) infection. At same time, we also discussed the effects of IDO expression on host's immune system. In partⅡ, the mammalian expression vector for murine IDO (IDO/pcDNA3.1/Zeo)was constructed.The recombinant plasmid was transfected into murine cell lines TC-1 cells from C57BL/6 mouse source to observe the cell's traits and effects on T lymphocyte. In partⅢ, we observed the characteristics of cell expressing IDO in vivo by a mouse model of cervix cancer using TC-1 cell transfected IDO/pcDNA3.1/Zeo vector and evaluate the effects on T lymphocyte. In a word, we explore the mechanisms of tumor escape associated with IDO to promote the development of means of eliminating tumor escape. Methods:Part I:Clinical study1 Indoleamine 2,3-Dioxygenase Expression in the CINI-III, Cervix cancer and the correlation analysis with high risk human papillomavirus infectionFrom April 2007 to April 2008,116 uterie cervical specimens and 18 metastatic lymph nodes specimens from patients with CINI-III and uterie cervical cancer by pathologically confirming and consistenting with enrollment criteria in the Third Affiliated Hospital of Kunming Medical College were evaluated for IDO expression by immunohistochemistry,20 normal cervical specimens and 20 normal lymph nodes specimens as control.The correlation between the IDO expression and HR-HPV infection was analyzed.2 The correlation between the IDO expression in cervix cancer tissue and the numbers of CD4+T lymphocyte,CD8+T lymphocyte, CD4~+ CD25~+ Foxp3~+ Tregs in peripheral blood.From June 2008 to October 2008, the patient consistenting with enrollment criteria in the Third Affiliated Hospital of Kunming Medical College were enrolled into this clinical trial.The patients were divided into three groups in according with pathological diagnosis:Normal cervix group(20 patients); CINIII group (20 patients) and cervix cancer group(20 patients).The specimen of cervix was obtained using the gynecatoptron for RT-PCR and Western blot to detect the IDOmRNA and IDO protein. The peripheral blood was obtained for analysis of numbers of CD4+,CD8+, CD4~+ CD25~+ Foxp3~+ Tregs.At same time, the correlation between the IDO expression in cervix cancer tissue and the numbers of CD4+T lymphocyte,CD8+T lymphocyte, CD4~+ CD25~+ Foxp3~+ Tregs in peripheral blood also was evaluated.Part II The experiment of cytobiology:the study of characteristics of cytobiology and effect on T lympholeukocytes with TC-1 cells expressing IDO1 Construct the expression vector of IDO (IDO/pcDNA3.1/Zeo)The IDO cDNA was amplified by RT-PCR from RAW 264.7 cells stimulated by Recombinant mouse interferon-y 200u/ml.The product of PCR was excised with HindⅢand BamHI and site in the pcDNA3.1/Zeo vector which was excised with HindⅢand BamHI.2 Obtain the stably transfected lines The IDO/pcDNA3.1/Zeo vector was transfected into TC-1 cell using lipofectamine 2000. Stably transfected lines were selected in 400u/ml zeocin.3 The experiment of cytobiology3.1 RT-PCR and Western blot detected the IDOmRNA and protein expression in IDO/pcDNA3.1/Zeo TC-cells3.2 The proliferation assay by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)1OOul/well of TC-1 cells, pcDNA3.1/Zeo TC-1 cells and IDO/pcDNA3.1/Zeo TC-1 cells were plated in 96 well plate at a density of 5×10~3/ml. After incubation 24 hours,48hours,96 hours,120 hours 168 hours and 216 hours, One plate was measured the absorbance by MTT.3.3 cell invasion assay300ul/well of TC-1 cells, pcDNA3.1/Zeo TC-1 cells and IDO/pcDNA3.1/Zeo TC-1 cells were plated in 24 well Matrigel invasion chambers at a density of.5×10~5 cells/ml.After incubation for 24 hours, the migranting cell on the lower surface were observed.3.4 The concentrations of tryptophan and kynurenine were measured by High-performance liquid chromatography.300ul/well of RPMI-1640/TC-1 cells, pcDNA3.1/Zeo TC-1 cells and IDO/pcDNA3.1/Zeo TC-1 cells were plated in 96 well plate at a density of.5×10~4 cells/ml.37℃,5% carbon dioxide incubation for 24 hours. The concentrations of tryptophan and kynurenine were measured by High-performance liquid chromatography.3.5 proliferation assay after mixed lymphocyte cocultures3.5.1 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) strained lymphocyte proliferation assay600ul/well of the blank control group (Splenocytes stained with CFSE),the control group(Splenocytes stained with CFSE+concanavalin A10ug/ml),the TC-1 group(Splenocytes stained with CFSE+concanavalin AlOug/ml+TC-1 cells), pcDNA3.1/Zeo TC-1 cells group(Splenocytes stained with CFSE+concanavalin A 10ug/ml+pcDNA3.1/Zeo TC-1 cells), IDO/pcDNA3.1/Zeo TC-1 cells group (Splenocytes stained with CFSE+concanavalin A 10ug/ml+IDO/pcDNA3.1/Zeo TC-lcells)were seeded into 24 well plate at a density of. l×lO~6 cells/ml.37℃,5% carbon dioxide incubation for 24 hours,48hours,72hours.The number of CD4+T lymphocyte and CD8+T lymphocyte and CFSE intensities were measured by flow cytometric analyses.3.5.2 CD4+CD25+Foxp3+Tregs proliferation assay after mixed lymphocyte cocultures600ul/well of the blank control group (Splenocytes),the control group(Splenocytes+ concanavalin A10ug/ml),the TC-1 group(Splenocytes+concanavalin A1Oug/ml+TC-1 cells), pcDNA3.1/Zeo TC-1 cells group(Splenocytes+concanavalin A 10ug/ml+pcDNA3.1/Zeo TC-1 cells), IDO/pcDNA3.1/Zeo TC-1 cells group(Splenocytes+concanavalin A 10ug/ml+ IDO/pcDNA3.1/Zeo TC-1 cells)were seeded into 24 well plate at a density of. 1×10~6 cells/ml. 37℃,5% carbon dioxide incubation for 24 hours.The number of CD4+CD25+Foxp3+Tregs were measured by flow cytometric analyses.3.6 T lymphocyte cytotoxicity assayThe CD8+T cells was obtained by positive magnetic isolation of CD8+T cells from tumor-bearing mice's spleen. 1OOul of the CD8+T cells as effector Cell and the 500cells/well of tumor cell as target Cell were seeded into 96 well plate with effector Cell/target Cell ration at 25:1,10:1,5:1. After 37℃,5% carbon dioxide incubation for 4 hours, the absorbance at 490 or 492nm was measured.Part III:Animal study.the IDO overexpression effect on immune system in mice mode of cervical cancer40 female C57BL/6 mice at 4-6 weeks of age and 18-22g weight were randomly divided to 4 groups(n=10), Physiologic saline, Physiologic saline and TC-cells, Physiologic saline and pcDNA3.1/Zeo TC-lcells, Physiologic saline and IDO/pcDNA3.1/Zeo TC-lcells were hypo inoculated into each mouse.The weight and the tumor volume were calculated. When the longest diameter reached to 1.0-1.5cm,mice were sacrificed.The weight of tumor was measured and the tumor tissue was evaluated for IDO expression by immunohistochemistry, RT-PCR and Western blot.The splenocyte of mice were used for mixed lymphocyte cultures of Part II. The peripheral blood was obtained for analysis of numbers of CD4+,CD8+, CD4+CD25+Foxp3+Tregs by flow cytometric analyses. The correlation between the IDO expression in tumor tissue and the numbers of CD4+T lymphocyte,CD8+T lymphocyte, CD4+CD25+Foxp3+Tregs in peripheral blood were analysized.Results:PartⅠ:Clinical study1 Indoleamine 2,3-Dioxygenase Expression in the CINI-Ⅲ, cervix cancer and the correlation analysis with high risk human papillomavirus infectionThe expression of IDO were not detected in normal cervix(20/20) and CINI(10/10).20% of CINⅡ,IDO expression were weakly positive(2/10) or negative(8/10,80%).61.5%(8/13) weakly positive expression,7.7%(1/13) positive expression and 30.8%(4/13) negative expression in CINⅢwere detected.The positive expression rate of IDO in cervical cancer stageⅠ—Ⅳwere 100%(83/83).In cervical cancer stage IA and IB, the positive expression rate of IDO were significantly greater than CINⅡ和CINⅢ(P<0.01). The positive expression rate of IDO in cervical cancer stageⅡA-ⅣB were significantly higher than IA and IB.IDO expression was associated with cervical cancer progression (OR=0.807, P< 0.01). IDO expression in tissue of lymph node metastases was significantly increase in lymph node tissue without metastases. The IDO expression was not associated with cervical cancer differentiation degree (OR=-0.139, P>0.05).The positive rates of HR-HPV were 20% in CINI(2/10),50% in CINⅡ(5/10),92.3% in CINⅢ(12/13),95.2%(79/83) in cervical cancer.The positive rates of HR-HPV and IDO were significantly higher in CINII-CINⅢand cervical cancer than in normal and CINI tissues (P<0.001),in cervical cancer than in CINⅡ-CINⅢ(P=0.007). The IDO expression in tissue with HR-HPV infection was significantly higher in tissue than the tissue without HR-HPV infection. Overexpression of IDO was positively correlated to HR-HPV infection in cervical cancer (rs=0.759, P＜O.001).2 The correlation between the IDO expression in cervix cancer tissue and the numbers of CD4+T lymphocyte,CD8+T lymphocyte, CD4+CD25+Foxp3+Tregs in peripheral bloodThe results of semiquantitative RT-PCR and Western blot show that the IDOmRNA and the IDO protein expression in cervix cancer tissue is significantly higher than in CINⅢ(P< 0.001,P<0.001) and normal cervix tissue (P<0.001,P<0.001).The IDOmRNA and the IDO protein expression in CINⅢtissue is significantly higher than in normal cervix tissue (P <0.001, P<0.001).The numbers of CD3~+T cells, CD4~+T cells, CD8~+T cells and CD4+/ CD8~+in peripheral blood of patients with cervix cancer were significantly lower than that of in patients with CINⅢ(P<0.001, P<0.001, P=0.005, P<0.001) or normal cervix (P <0,001, P<0.001, P=0.026, P<0.001).But there was not different in the women with normal cervix and the patients with CINⅢ. (P=0.288, P=0.658, P=0.524, P=0.964).The number of CD4+CD25+Foxp3+Tregs in patients with cervix cancer was significantly higher than in patients with CINⅢ(P<0.001) or normal cervix (P<0.001).The IDOmRNA and protein expression in normal cervix tissue inversely correlated with CD3+Tcells (r=-0.537, P=0.015; r=-0455 P=0.044) and CD4~+/CD8~+(r=-0.654, P=0.002, r=-0.684, P=0.001) and directely correlated with CD4+CD25+Foxp3+Tregs (r=0.487, P=0.029, r=0.514,:P=0.020). The IDOmRNA and protein expression in CINⅢinversely correlated with CD3+Tcells (r=-0.783, P<0.001; r=-0.768, P<0.001) and CD4~+/CD8+ (r=-0.778, P< 0.001, r=-0.771, P< 0.001) and directely correlated with CD4+CD25+Foxp3+Tregs (r=0.841,P<0.001, r=0.850, P<0.001). The IDOmRNA and protein expression in cervix cancer inversely correlated with CD3+Tcells (r=-O.881,P <0.001; r=-0.919, P<0.001) andCD4+/CD8+ Tcells (r=-0.870, P<0.001, r=-0.733, P<0.001) and directely correlated with CD4+CD25+Foxp3+Tregs (r=0.487, P=0.029, r=0.514, P=0.020).PartⅡ:The experiment of cytobiologyThe study of characteristics of cytobiology and effect on T lympholeukocytes with TC-1 cells expressing IDO1 the expression vector of IDO (IDO/pcDNA3.1/Zeo) excised with HindⅢand BamHI and the 1.2kb band was observed.The the expression vector of IDO (IDO/pcDNA3.1/Zeo) was conformed by sequence analysis.2 RT-PCR and Western blot detected the IDOmRNA and protein expression in IDO/pcDNA3.1/Zeo TC-cellsthe IDO/pcDNA3.1/Zeo TC-cells expressed the IDOmRNA and protein because the IDO cDNA band in 233bp and IDO protein band in 45kd were detected.There were no IDOmRNA expression in the TC-1 cells and pcDNA3.1/Zeo TC-cells. 3 The proliferation assay by MTTThe proliferation were not diferent in TC-1 cells,the pcDNA3.1/Zeo+cells and the IDO/pcDNA3.1/Zeo+TC-1 cells After incubation 24 hours (F=0.47, P>0.05),48hours (F=3.064, P>0.05),96 hours (F=1.55 P>0.05),120 hours (F=0.744, P>0.05)and 216 hours (F=0.110P>0.05).4 cell invasion assayThe are 29.688±5.437 invasive cells/HP in group of IDO/pcDNA3.1/Zeo+TC-1 cells, 16.688±2.024 invasive cells/HP in group of pcDNA3.1/ZeoTC-1 cell and 18.875±3.845 invasive cells/HP in group of TC-1 cell.The value of absorbance at 560nm were 0.979±0.024 in group of IDO/pcDNA3.1/Zeo+TC-1 cells,0.745±0.127 invasive cells/HP in pcDNA3.1/Zeo+TC-1 cell and 0.727±0.045 invasive cells/HP in group of TC-1 cell.The invasive cells/HP and the value of absorbance at 560nm in group of IDO/pcDNA3.1/Zeo+TC-1 cells was significantly higher than in groups of pcDNA3.1/Zeo+TC-1 cells(P<0.001,P<0.01) and TC-1 cells(P<0.001,P<0.01).There was no difference in group of pcDNA3.1/Zeo+TC-1 cells and group of TC-1 cells (P>0.05, P>0.05).5 The concentrations of tryptophan and kynurenineIn group of IDO/pcDNA3.1/Zeo TC-1 cells,the concentrations of tryptophan was significantly lower than in RPMI-1640 medium (P<0.001), groups of pcDNA3.1/Zeo TC-1 cells (P<0.001, P<0.001) andTC-1 cells (P<0.001, P<0.001).The concentrations of kynurenine was significantly higher than that of other groups (P<0.001, P<0.001,,P< 0.001). There was no difference in group of pcDNA3.1/ZeoTC-1 cells and group of TC-1 cells (P=0.949, P=0.803)6 CFSE strained lymphocyte proliferation assayAfter cocultured with spenocytes for 24-72 hours, The CD4+T cells and CD8+T cells numbers of blank control group, control group, TC-cells group and pcDNA3.1/ZeoTC-1 cells group significantly increased over the same period when cultured with IDO/pcDNA3.1/Zeo TC-1 cells group (P<0.01).The munbers of CD4~+T cells and CD8+T cells in control group, TC-cells group and pcDNA3.1/ZeoTC-1 cells group significantly higher than that of blank control group (P<0.01)When concultured for 72 hours, CFSE straining profiles revealed that the CD4+T cells and CD8~+T cells had divided 2-3 times in blank control groups. the CD4+T cells in IDO/pcDNA3.1/Zeo TC-1 cells group revealed had divided 1-2 times.The CFSE intensities of CD8+T cells was similar to the undivided cell before the cocultrue in 2 well samples.One sample revealed that CD8+T cells had divided one time. The control group, TC-1 cells group and pcDNA3.1/Zeo TC-1 cells group revealed had divided at least 4 times.7 CD4+CD25+Foxp3+Tregs proliferation assay after mixed lymphocyte culturesThe numbers of CD4+CD25+Foxp3+Tregs in IDO/pcDNA3.1/Zeo TC-1 cells group was significantly increaseing (P<0.01) but no differece in other groups (P>0.05).8 lymphocyte cytotoxicity assayThe purity of CD8+T cells'was 91.5% by flow cytometry analysis.CD8+T cells cocultured with TC-1 cells,or pcDNA3.1/Zeo TC-1 cells, or IDO/pcDNA3.1/Zeo TC-1 cells for 4 hours, the cytotoxicity was 18.189±0.514% in TC-1 cells group,18.879±0.397% in pcDNA3.1/Zeo TC-1 cells group,and only 9.412±0.398% in IDO/pcDNA3.1/ZeoTC-1 cells groups when the effector Cell/target Cell ration at 10:1. (P <0.01, P<0.01).Part III:Animal study1 The general appearance90%(9/10) of tumors in TC-1 cells group,80%(8/10) in pcDNA3.1/ZeoTC-1 cells groups and 100%(10/10) in IDO/pcDNA3.1/ZeoTC-1 cells groups were observed at 4 days after inoculation.There were no tumor was observed in the blank control group.The growth rate and the weight of tumor in IDO/pcDNA3.1/ZeoTC-1 cells groups was significantly enhanced from day 6 after the tumor emerged (P<0.01, P<0.01).The weight of mice was significantly decreasing in IDO/pcDNA3.1/ZeoTC-1 cells groups (P<0.001).2 Evaluate the IDO expression in tumor tissue by immunohistochemistryThe expression of IDO were detected in all tumor tissue. The positive rate of IDO expression in IDO/pcDNA3.1/ZeoTC-1 cells groups was higher than in pcDNA3.1/ZeoTC-1 cells groups (t=5.7266,P<0.001) and in TC-1 cells groups (t=0.5074, P>0.05). 3. The numbers of CD3+T cells, CD4+T cells,CD8~+T cells, CD4+CD25+Foxp3+Tregs in peripheral blood by flow cytometric analysesCompared with blank control group, the numbers of CD3+T cells, CD4+T cells,CD8~+T cells in TC-1 cells groups, pcDNA3.1/ZeoTC-1 cells groups and IDO/pcDNA3.1/ZeoTC-1 cells groups were sinificantly decreasing and The numbers of CD4+CD25+Foxp3+Tregs were sinificantly increasing. (P<0.05, P<0.01, P<0.001).There are no diference in TC-1 cells groups and pcDNA3.1/ZeoTC-1 cells groups The numbers of CD3+T cells, CD4+T cells,CD8~+T cells significantly decreased and the numbers of CD4+CD25+Foxp3+Tregs significantly increased in IDO/pcDNA3.1/ZeoTC-1 cells groups.4 The IDO mRNA and protein expression in tumor using semiquantitative RT-PCR and Western blot.higher levels of IDOmRNA and protein were expressed by the tumor tissue of IDO/pcDNA3.1/ZeoTC-1 cells groups compare with other groups. (P<0.01, P<0.001). There were no difference in the tumor tissue of pcDNA3.1/ZeoTC-1 cells groups and TC-1 cells groups.5 The correlation between the IDO expression in tumor tissue and the numbers of CD4+T lymphocyte,CD8+T lymphocyte, CD4+CD25+Foxp3+Tregs in peripheral blood.The IDOmRNA and protein expressing in tumor tissue of IDO/pcDNA3.1/ZeoTC-1 cells groups were negative correlation with CD3+T cell(r=-0.851, P=0.002; r=-0.792, P=0.006), CD4~+/CD8~+(r=-0.809, P=0.005, r=-0.806, P=0.005) and positive correlation with CD4+CD25+Foxp3+Tregs 0=0.886, P=0.001, r=0.872, P=0.001)Conclusions:1 IDO high expression in cervical cancer tissue closely linked to cervical cancer.2 IDO expression was correlated with clinical stage and of cervical cancer; progression of disease and HR-HPV infection,but was not correlated with differentiation degree of cervical cancer.3 IDO expression was correlated with lymph node metastasis.4 Cervical cancer cells expressing IDO can suppress the proliferation and immunocompetence of T cells.The mechanisms could be correlated with tryptophan metabolism.5 Concanavalin A could not be the activating agent induced CD4~+ CD25~+ Foxp3~+ Tregs to expand,but IDO could be.6 Cervix cancer cells expressing IDO to expand the numbers of CD4~+ CD25~+ Foxp3~+ Tregs coul be one of factors leading to immune escape.7 The tumor cells expressing IDO revealed enhanced invasion abilities but its proliferation competence wasn't be influenced.14 The invasion abilities of tumor cells expressing IDO would be associated with tryptophan catabolism.15 IDO and CD4+CD25+Foxp3+Tregs construct the systems of positive feedback which IDO is the hinge.The expression of IDO be induced by CD4+ CD25+ Foxp3+ Tregs. Conversely,IDO promtes the differentiation of CD4+CD25+Foxp3+Tregs.IDO would be one of important new therapeutic targets in the future.