1. The Expansion And Conversion Of CD4~+CD25~+ Regulatory T Cell 2. The High Frequect Lack Of PDCD4 Expression In Human Gliomas

ObjectiveRegulatory T cells(Tregs)are one of hot topics in immunology research for the past a few years.With the further research of regulatory T cells,it has been known that Tregs exert a very important protective role in maintaining immune tolerance, controlling autoimmune disease and transplantation rejection reaction.But in tumor, they suppress the anti-tumor effect of immune system.Among regulatory T cells, CD4⁺CD25⁺Treg cells are the most studied population.It has been known that CD4⁺CD25⁺Treg cells have two sub-sets:naturally occurring CD4⁺CD25⁺Treg cells (nTregs)and inducible CD4⁺CD25⁺Treg cells(iTregs).The nTreg cells are from thymus and account for approximately 5-10%of CD4⁺T cells.The major markers for the Treg include CTLA-4,GITR and newly-discovered transcription factor Foxp3,etc. They could suppress self-reactive T cells through cell-cell contact.The iTreg cells ale converted from CD4⁺CD25^-T cells under certain conditions(e.g.TGF-β,IL-10,etc). They have the same surface markers and suppressive function as nTregs have.But they differ from nTregs in that they exert suppressive function through cytokine.Recently,the discovery of CD4⁺CD25⁺Treg cells specific marker-Foxp3 and the TGF-βassisted in vitro conversion of CD4⁺CD25^-T cells into the CD4⁺CD25⁺Treg cells which have the suppressive function have greatly promoted development of CD4⁺CD25⁺Treg cells research.It sheds light into treating autoimmune diseases and controlling transplantation rejection reaction through using CD4⁺CD25⁺Treg cells. Several prominent immunologists have predicted that the most valuable clinical application of CD4⁺CD25⁺Treg cells is in managing Graft-versus-Host Disease (GVHD)and transplantation rejection reaction.The bottleneck of administering the CD4⁺CD25⁺Treg cells for disease treatment is the insufficiency of Treg cells.The CD4⁺CD25⁺Treg cells account for 5-10%of peripheral CD4⁺T cells in mice but less than 5%in human beings.In order to treat diseases,the amount of CD4⁺CD25⁺Treg cells from one donor is insufficient. Therefore,it becomes imperative to find ways obtaining enough CD4⁺CD25⁺Treg cells.We already knew that co-stimulatory molecule plays an important role in T cells activation and proliferation.In recent years,we found that 4-1BB also expresses on the surface of CD4⁺CD25⁺Treg cells.However,the function of 4-1BB on CD4⁺CD25⁺ Treg cells remains unknown.Hence,one of the purposes of this research is to identify 4-1BB's role in the proliferation of CD4⁺CD25⁺Treg cells.The research not only explores new ways to provide sufficient amount of CD4⁺CD25⁺Treg cells,but offers theoretical basis for administering CD4⁺CD25⁺Treg cells in GVHD.CD4⁺CD25⁺Treg cells play an important protective role in autoimmune disease and transplantation rejection reaction.But in tumor,they depress anti-tumor capability of the body.In recent years,it has been known that lymphocytes in the vicinity of tumor have not only effector CD8⁺T cells or CD4⁺T cells,but also plenty of CD4⁺CD25⁺Treg cells.These CD4⁺CD25⁺Treg cells are involved in tumor immunity escape.Increased numbers of CD4⁺CD25⁺Treg cells are found in cancer patients' peripheral blood,tumor infiltrating lymph tissue(TIL),draining lymph nodes and ascites.However,origin of these cells is unknown.To identify the origin of CD4⁺CD25⁺Treg cells is useful for exploring to eliminate these cells and establish effective tumor immunology therapy modality.Therefore,the second purpose of the research into study the correlation between increased CD4⁺CD25⁺Treg cells and tumor size and explore the mechanism the CD4⁺CD25⁺Treg cells elevation in tumor growth using H22 hepatic carcinoma-bearing BALB/c mice as model.Methods1.The expansion effect of 4-1BB monoclonal antibody on naturally occurring CD4⁺CD25⁺Treg cells(1)To isolate and purify CD4⁺CD25^-T cells from mice spleen and CD4⁺CD25⁺Treg cells by using mice CD4⁺CD25⁺T cell isolation Kit.(2)In vitro,detect the effect of 4-1BB monoclonal antibody on proliferation of CD4⁺CD25⁺Treg cells was detected using 3H-TdR incorporation.(3)To detect the effect of 4-1BB monoclonal antibody on cytokine production of CD4⁺CD25⁺Treg cells using ELISA.(4)To detect CD4⁺CD25⁺Treg cells phenotypes and Foxp3 expression using RT-PCR and FCM.(5)In vitro,the inhibitive effect of 4-1BB mAb-expanded CD4+CD25+Treg cells was detected using 3H-TdR incorporation.2.Induction of hepatic carcinoma on CD4⁺CD25⁺Treg cells(1)To prepare H22 hepatic carcinoma BALB/c mice.(2)To detect the change of CD4⁺CD25⁺T cells numbers in tumor bearing mice using FCM.(3)To detect Foxp3 expression in H22 hepatic carcinoma bearing BALB/c mice spleen and lymph nodes using RT-PCR and FCM.(4)In vitro,detect the inhibitive effect of tumor bearing mice-originated CD4⁺CD25⁺T cells using 3H-TdR incorporation.(5)To infuse tumor bearing mice-originated CD4⁺CD25⁺T cells and CD4⁺CD25^-T cells to tumor bearing nude mice,then detect inhibitive function of CD4⁺CD25⁺T cells to CD4+CD25^-T cells anti-cancer effect.(6)To detect expression of cytokine TGF-βin plasma of tumor bearing SCID mice using ELISA.Resultsâ… .4-1BB mAb and CD4⁺CD25⁺Treg cells expansion.1.Purification of CD4⁺CD25^-T cells and CD4⁺CD25⁺T cells.The results from FCM showed that purification of CD4⁺CD25^-T cells could reach 95%and purification of CD4⁺CD25⁺T cells could reach 90%.2.The expansion effect of 4-1BB mAb on CD4⁺CD25⁺Treg cells and its dependence on IL-2.The results from T cell proliferation showed that without exogenous IL-2 or APCs cells,CD3 TCR signal in presence or absence of 4-1BB co-stimulatory signal can't expand CD4⁺CD25⁺ Treg cells.However,with co-existence of low concentration cytokine IL-2,4-1BB could synergistically expand CD4⁺CD25⁺ Treg cells with the presence of CD3 antibody.With the co-existence of APCs cells but no exogenous IL-2 cells,the same effect could also be observed.Furthermore,after the action of IL-2 was neutralized by ani-IL-2 mAb,4-1BB mAb can't expand CD4⁺CD25⁺ Treg cells even with the presence of APCs cells.These results demonstrated that IL-2 was necessary for expansion of CD4⁺CD25⁺ Treg cells stimulated by 4-1BB Ab.3.The characteristics of 4-1BB mAb expanded CD4⁺CD25⁺ Treg cells.3.1.4-1BB mAb expanded CD4⁺CD25⁺ Treg cells still express Foxp3.The Foxp3 is a specific marker for regulatory T cells.In order to confirm whether 4-1BB mAb-expanded CD4⁺CD25⁺ T cells still have regulatory characteristics,we tested Foxp3 expression on RNA and protein level by using RT-PCR,real time PCR and FCM.The RT-PCR results showed that 4-1BB mAb-expanded CD4⁺CD25⁺Treg cell still express high level of Foxp3.Real time PCR results showed that 4-1BB mAb-expanded CD4⁺CD25⁺Treg cells have slightly elevated levels of Foxp3 mRNA, comparing with control group.FCM results indicated that Foxp3 expression had no significant difference between 4-1BB mAb expanded CD4⁺CD25⁺Treg cells and CD4⁺CD25⁺Treg cells in control group.The results suggested that 4-1BB mAb expanded-CD4⁺CD25⁺Treg cells still are regulatory T cells features. 3.2 4-1BB mAb expanded CD4⁺CD25⁺Treg cells produce low level of IL-2 and high level of IFN-γ.The detection of ELISA showed that although 4-1BB mAb expanded CD4⁺CD25⁺T cells produce high level of IL-2,4-1BB mAb expanded CD4⁺CD25⁺T reg cells only produce low level of IL-2.The difference is that 4-1BB mAb expanded CD4⁺CD25⁺Treg cells produce high level of IFN-γ.The significance remains unknown.3.3.4-1BB mAb expanded-CD4⁺CD25⁺Treg cells remain inhibitive function.Use CD3 antibody and APCs cells to stimulate newly isolated BALB/c CD4⁺CD25⁺T cells,then cultured with 4-1BB mAb expanded CD4⁺CD25⁺Treg cells. After 3 days,use ³H-TdR incorporation assay to detect cell proliferation.Inhibition rate of post expansion CD4⁺CD25⁺Treg cells could reach 35.36%,which has no significant difference from newly isolated CD4⁺CD25⁺Treg cells.â…¡.Induction of hepatic carcinoma on CD4⁺CD25⁺Treg cells.1.The number of CD4⁺CD25⁺T cells in the spleen and lymph nodes of H22 hepatic carcinoma mice is elevated.BALB/c mice were inoculated subcutaneously with H22 hepatic carcinoma cells. 4-5 weeks later,appeared with tumor.Use FCM to detect spleen,lymph nodes and draining lymph nodes cell surface marker CD4 and CD25 expression.The results show that comparing with no tumor bearing BALB/c mice,the percentage of CD4⁺CD25⁺T/CD4⁺T increases significantly in H22 hepatic carcinoma bearing mice spleen,lymph nodes and draining lymph nodes.Spleen:17.28+0.06%vs. 11.08±0.04%,P＜0.05;draining lymph nodes:18.8±0.06%vs.9.5±0.03%,P＜0.01; non-draining lymph nodes:16.28±0.02%vs.9.5±0.03%,P＜0.01.Draining lymph nodes have slightly higher percentage than non-draining lymph nodes,but there is no statistically significant difference(P＞0.05).2.The CD4⁺CD25⁺T cells number is positively correlated with tumor size.Prepare tumor bearing mice from H22 hepatic carcinoma with different concentration and measure tumor size every other day.After 3-4 weeks,kill mice, isolate spleen,lymph nodes,draining lymph nodes and then isolate cells.Use FCM to detect CD4,CD25 expression on cell surface.The results show that with the increase of tumor sizes in H22 hepatic carcinoma bearing mice,the percentage of spleen CD4⁺CD25⁺T/CD4⁺T cells increases,which indicates CD4⁺CD25⁺T count will benefit tumor growth.3.H22 hepatic carcinoma induced Treg specific marker-Foxp3 expression.RT-PCR electrophoresis results show that comparing with no tumor bearing BALB/c mice,Foxp3 expression increases in the spleen and lymph nodes(including draining lymph nodes and non-draining lymph nodes)of H22 tumor beating mice. Further test of Foxp3 protein expression through FCM shows that transcript factor Foxp3 protein expression in H22 tumor beating mice is significantly higher than that of the control group BALB/c mice.4.In vitro,H22 tumor bearing mice CD4⁺CD25⁺T cells are anergy and can inhibit CD4⁺CD25⁺T cells proliferation.Isolated H22 tumor bearing mice spleen CD4⁺CD25⁺T cells,stimulate with CD3 mAb and APCs cells,culture for 3 days under 37℃,5%CO2;3H-TdR incorporation proliferation results show that CD4⁺CD25⁺T cells from H22 tumor bearing mice don't proliferate.The suppressive assay shows that the CD4⁺CD25⁺T cells could inhibit the proliferation of the CD4⁺CD25^-T cells.The ration of inhibition is 34.59%.5.In vitro,the increased CD4⁺CD25⁺T cells from the tumor bearing BALB/c mice can inhibit CD4⁺CD25^-T cells anti-tumor effect.Infuse purified CD4⁺CD25^-T cells and CD4⁺CD25⁺T cells into H22 tumor bearing nude mice at a ratio of CD4⁺CD25⁺T:CD4⁺CD25^-T=2:1.After 9 days,tumor size in nude mice only received CD4⁺CD25^-T cells infusion is significantly smaller than the other two groups,tumor size of nude mice received mixture of CD4⁺CD25^-T cells CD4⁺CD25⁺T cells is smaller than H22 hepatic carcinoma nude mice not receiving any infusion.6.In H22 tumor-bearing mice and SCID mice,CD4⁺CD25^-T cells can convert into CD4⁺CD25⁺Foxp3⁺T cells.In order to identify whether elevated CD4⁺CD25⁺T cells from tumor bearing mice are converted from CD4⁺CD25^-T cells,we use the purified CD4⁺CD25^-T cells of regular BALB/c mice to inject to tumor bearing nude mice or SCID mice.After 5-10 days,isolate tumor bearing nude mice spleen,lymph nodes,and use FCM to detect presence of CD4⁺CD25⁺Treg.The results show that most infused CD4⁺CD25^-T cells in tumor bearing nude mice or SCID mice converted into CD4⁺CD25⁺T cells.In order to further test whether these cells are regulatory T cells,we adopted RT-PCR to detect H22 tumor bearing nude mice Foxp3 gene expression.Comparing with H22 tumor bearing nude mice not receiving CD4⁺CD25^-T cells,the ones injected with CD4⁺CD25^-T cells have elevated Foxp3 gene expression in spleen and lymph nodes. FCM shows that in tumor bearing SCID mice,these CD4⁺CD25⁺T cells have high expressed Foxp3 protein.FCM shows that CD4⁺CD25⁺Foxp3⁺T cells account for 80-85%of CD4⁺T cells.The aforementioned results demonstrate that H22 hepatic carcinoma can induce conversion of CD4⁺CD25^-T cells into CD4⁺CD25⁺Foxp3Treg cells.This is could be one of the reasons tumor bearing mice have elevated levels of regulatory T cells.7.The TGF-βcytokine expression increased in the plasma of the H22 hepatic carcinoma and this could be one of the reasons of tumor induced CD4⁺CD25⁺Treg cells production.We already knew that in vitro,TGF-βcan induce conversion of CD4⁺CD25^-T cells into CD4⁺CD25⁺Treg cells.Whether TGF-βplayed a role in CD4⁺CD25^-T conversion into CD4⁺CD25⁺Foxp3^Treg cells in H22 tumor bearing mice is unknown.ELISA test shows that H22 hepatic carcinoma supernatant has higher levels of TGF-βcytokines (1.2ug/ml).Also,tumor bearing SCID mice plasma have large amounts of TGF-β,this indicates that H22 tumor bearing mice can produce high level of TGF-β.This could be one of the reasons tumor induced CD4⁺CD25⁺Treg cells,but more research needs to be done.8.In vivo,TGF-βcytokine is involved in CD4⁺CD25⁺Foxp3⁺Treg conversion.Tumor-bearing Rag1^-/-mice were injected with CD4⁺CD25^-T cells,which comes from T cell specific TGF-βRI knockout mice.After 15 days,CD4,CD25,Foxp3 molecules expression in mice spleen,LN and draining LN were measured by FCM. The results show that the conversion of CD4⁺CD25^-T cells into CD4⁺CD25⁺Foxp3⁺Treg Cells is much less in tumor-bearing Rag1^-/-mice injected knockout mice CD4⁺CD25^-T cells than in Tumor-bearing Rag1^-/-mice injected the wild type CD4⁺CD25^-T cells. Conclusionâ… .The effect of 4-1BB mAb on expansion of CD4⁺CD25⁺Treg cells.1.In vitro,the expansion of natural CD4⁺CD25⁺Treg cells by agonistic anti-4-1BB antibody needs APC or exogenous IL-2.2.The expanded CD4⁺CD25⁺Treg by 4-1BB mAb produce a few IL-2,but large amounts of IFN-γ3.4-1BB mAb-expanded CD4⁺CD25⁺Treg cells still express Foxp3.4.The CD4⁺CD25⁺Treg cells expanded by anti-4-1BB antibody remain suppressive activity to CD4⁺CD25^-T cells.â…¡.Hepatic carcinoma induces CD4⁺CD25⁺Treg cells.1.The number of CD4⁺CD25⁺T cells in lymph nodes and spleen of H22 tumor-bearing mice is higher than that of tumor free mice.The number of CD4⁺CD25⁺T cells in draining lymph nodes is higher than that in non-draining lymph nodes.2.The number of CD4⁺CD25⁺T/CD4⁺T cells is positively related to tumor size in H22 tumor-bearing mice.3.Transcript factor Foxp3 expression increases in the spleen and lymph nodes of H22 hepatic carcinoma mice. 4.In vitro,the CD4⁺CD25⁺T cells from H22 tumor bearing-mice are anergy and can inhibit proliferation of conventional CD⁺CD25T cells.5.In vivo,the CD4⁺CD25⁺T cells can inhibit the anti-tumor effect of conventional CD4+^CD25^-T cells in tumor bearing-nude mice.6.The proliferation of H22 tumor cells can induce the conversion of CD4⁺CD25^-T into CD4⁺CD25⁺foxp3⁺T cells in SCID mice.7.The H22 cells can secret high level of TGF-βin condition of culture or in tumor-bearing SCID mice.8.In vivo,TGF-βcytokine is involved in CD4⁺CD25⁺Foxp3⁺Treg conversion.Originalityâ… .4-1BB mAb affect CD4⁺CD25⁺Treg cells proliferation.1.We discovered for the first time that 4-1BB mAb-activated costimulatory pathway can effectively expand CD4⁺CD25⁺Treg cells in dependent manner of IL-2.2.We have demonstrated that 4-1BB mAb-expanded CD4⁺CD25⁺Treg cells maintain regulatory T calls features,expression of Foxp3,production of low levels of IL-2 and immune suppression to conventional T cells.â…¡.Hepatic carcinoma can induce CD4⁺CD25⁺Treg cells.1.We have found that the proliferation of H22 hepatocellular carcinoma can increase the number of CD4⁺CD25⁺T cells in spleen and lymph nodes and the elevated number of CD4⁺CD25⁺T cells is positively correlated with size of tumor growth in mice.2.The proliferation of H22 hepaocellular carcinoma in SCID mice can induce conversion of CD4⁺CD25^-T cells into CD4⁺CD25⁺Foxp3⁺Treg cells,which is one reason for increase of CD4⁺CD25⁺Tregs cells in tumor-bearing mice.3.Be confirmed in vivo,TGF-βreleased by tumor is involved in CD4⁺CD25⁺Foxp3⁺Treg cells conversion.Limitations of this study1.Although it has been found in this research that 4-1BB mAb can stimulate CD4⁺CD25⁺Treg cells to produce high level of IFN-γ,its mechanism and significance have not been studied.2.The function of TGF-βin conversion of CD4⁺CD25⁺Treg cells by H22 tumor cells needs to be studied further. ObjectiveProgrammed cell death 4,PDCD4,was cloned Shibahara et al in from mouse cDNA.The expression of PDCD4 was up-regulated during apoptosis in all cell lines tested.Programmed cell death 4(PDCD4)is a translation factor that binds to eIF4A Recent evidence suggests that PDCD4 acts a tumor suppressor oncogene and might represent a promising target for future antitumor therapy.There is e few reports about the function of PDCD4 gene in human tumor progress.Recently,it is known that the tumor cells which derive from kidney,lung and colon express less PDCD4 or lack.In the original lung cancer,the frequency of PDCD4 lack reaches to 83%,and there is a relationship between PDCD4 lack and tumor differentiation or the patient's prognosis.The same results had been found in the pancreatic tumor.These demonstrate that PDCD4 is a suppressor oncogene. However,It is unknown that the expression character of PDCD4 gene in other organ tumor and whether it is significant in clinical.Glioma is one of the common tumors in central nervous system,A half of the gliomas are histological malignant.Although the operation,radiotherapy and chemotherapy have been used for cancer treatments,the prognosis of patients with glioma is bad.It has been reported that five of six cell lines from central nervous system,lacked the PDCD4 expression.However,the situation of PDCD4 in primary nervous tumors is unknown.The objective of this research clarifies the character of PDCD4 expression in gliomes and clinical significance.The results maybe provide the new strategy for the gliomas therapy.Methods1.Glioma specimens were obtained from 30 patients with tumor aged between 40 and 60 years(mean 50 years)who underwent operations at the Department of Neurosurgery in QiLu Hospital of Shandong University from March 2005 to March 2006.No patient had received adjuvant immunosuppressive treatment including radiotherapy or chemotherapy prior to surgery.The specimens were immediately frozen in liquid nitrogen after surgical tissue removal,and then kept at -80℃.The three normal glial tissues were all acquired from tumor adjacent tissues.2.Total RNAs and protein were extracted from 30 glioma specimens,3 normal tissue specimens and glioma cell line(U251)cells using a modified TRIzol(?)one-step extraction method.3.The PDCD4 mRNA and protein expression in 30 cases of glioma specimens,3 normal tissue specimens and glioma cell line cells were detected by RT-PCR,Western blot and immunohistochemistry.4.The relationship between the PDCD4 expression and the pathological characters was analyzed by staticallyResults 1.Lacked or Decreased expression of PDCD4 mRNA in gliomas.To study role of PDCD4 in the gliomas,we firstly examined the PDCD4 expression at RNA level by semi-quantitative RT-PCR according to normalized OD value of PDCD4 mRNA expression toβ-actin:-,no detection;+,0.01 to 0.49;++,0.5 to 0.99;+++,1.0 to 1.99;++++,=2.00.As illustrated in Fig.1,all of three normal brain tissues adjacent to tumor lesion expressed high level of PDCD4 mRNA, whereas the most of gliomas expressed reduced or lacked PDCD4 mRNA.In 30 glioma specimens,14(47%,14/30)lost PDCD4 mRNA expression.In the remaining 16 glioma samples,12(40%,12/30)appeared differently reduction in PDCD4 mRNA expression compared with three normal tissue controls.For example,PDCD4s were lost in the glioma sample 2,3,7 and distinctly reduced in the glioma sample 4,5,6; while glioma sample 1 was almost identical with the normal tissue control.U251 is cell line originated from glioma.It has reported that the cell line almost loses expression of PDCD4 mRNA.Our result was identical with the previous report.2.High frequent loss of PDCD4 protein expression in gliomas.Based on above studies,we further examined the expression of PDCD4 protein by Western blot.The data from Western blot experiment showed that the loss of PDCD4 proteins was more than that of PDCD4 mRNA.In 30 cases of the gliomas,23 cases(77%)exhibited no relevant PDCD4 bands and other 6 glioma cases showed weak PDCD4 bands,compared with normal brain tissues adjacent to tumor,which showed strong PDCD4 expression.Only sample 1,4,6 had slight expression.While other samples,e.g.,sample 2,3,5,7 were lost in the protein level.Note worthily, some gliomas such as sample 5 expressed PDCD4 mRNA but not PDCD4 protein. These suggested that loss of PDCD4 expression in certain gliomas might occur after mRNA transcription.3.The high frequent loss of PDCD4 protein expression in gliomas was confirmed further by immunohistochemistry.Immunohistochemisry analysis showed that the PDCD4 protein expression in the tumor tissue sections were similar to those from Western blot analysis.The tumor adjacent tissue such as case 24 showed very strong PDCD4 expression,while its paired tumor tissue exhibited decreased PDCD4 staining.In certain gliomas,such as case 29,even there was almost no relevant PDCD4 staining.In addition,it showed that the positive PDCD4 staining localized in cytoplasm.4.The loss of PDCD4 in gliomas had no signification relationships with tumor grade or histological typeTo explore the significance of lacked PDCD4 protein in gliomas,we further analyzed the relationship between loss of PDCD4 proteins and clinic pathological parameters.As expected,there was no significant correlation of lacked PDCD4 expression with sex,age,and tumor size.Unexpectedly there was also no significant relationship between loss of PDCD4 expression and tumor grade or histological type. We are following up the relationship between loss of PDCD4 expression and the prognosis of the gliomas patientsConclusion 1.There are a lacked or decreased expression of PDCD4 mRNA in gliomas,the rate of the lack of PDCD4 expression to 47%.2.High frequent loss of PDCD4 protein expression is found in gliomas,the rate of the lack of PDCD4 expression to 77%.3.The loss of PDCD4 in gliomas had no signification relationships with tumor grade or histological type.OriginalityThis is the first time to discover that high frequent loss of PDCD4 protein expression in gliomas,and to research the relationship between PDCD4 expression and tumor grade or histological type.It provides theoretical basis for PDCD4 on glioma pathogenesis and its inhibitive mechanism on tumor.Limitation1.The more cases are need for next step study.2.With limited follow up time,the correlation between PDCD4 expression and prognosis of patients with glioma has not been finished.We are continuing this follow up study.