Experimental Study On The Antitumor Effects Of CTL Derived From Umbilical Cord Blood Na?ve T Cells Activated By EpCAM Loaded DC

In recent years,the conventional treatment of malignant tumors,including surgery,radiotherapy and chemotherapy,has encountered choke point.With improvement of the understanding of the relationship between development of tumor and organism immunity,biotherapy has become a new way to fight tumour.Biotherapy includes cell therapy and non cellular therapy(gene therapy,antiangiogenic therapy,small molecule targeted drug therapy,monoclonal antibody therapy,peptide or protein vaccines,gene vaccines,etc.).Cell immunotherapy is to apply tumor patients with anti-tumor activity of the immune cells,directly kill the tumor cells or stimulate the body anti-tumor immune response,so as to treat cancers;this process is called tumor adoptive cell therapy(ACT).The core of cellular immunotherapy is to induce T cell activation and to recognize tumor antigens,resulting in tumor killing.There are two main modes,one is DC-induced T cell immunity,mainly through DC that loading the corresponding antigen to presente the antigen information to the T cells,so that T cells activated and can specifically identify and kill tumor cells;Second,T cells Engineering preparation,mainly through T cell gene transfection or gene editing,so that T cells can specifically identify and kill tumor cells.And DC-induced T cell immunity is an important research direction of cellular immunotherapy.At present,peripheral blood extraction of mononuclear cells to amplify and activate is widely used in clinical treatment,with process of the application of autologous or allogeneic tumor cells to prepare antigens,and then the preparation of antigen-specific killer cells,apply patients with recurrent transfusion therapy.This method has achieved a certain effect.However,the tumor patients' condition,T cell function status,antigenspecificity,tumor microenvironment and other issues limit its clinical application and reduce the clinical efficacy.First of all,the current clinical application of DC cells and T cells is from peripheral blood mononuclear cells collected.The tumor "immunosuppressive microenvironment" leads to autologous DC and T cell dysfunction and the number of insufficient,advanced cancer patients can not tolerate a large number of peripheral blood mononuclear cells in the collection and other issues,resulting in the defects of DC and T cells in function and the number,and limited the industrialization.Recent studies have shown that effector cells prepared by different T cell subpopulations have different anti-tumor effects.Among them,na?ve T cell subsets had stronger anti-tumor ability than memory T cells.T lymphocytes derived from umbilical cord blood have stronger amplification and activation ability than peripheral blood T lymphocytes,and are widely used in hematological malignancies,and also show strong killing ability for solid tumors.However,there is no study of the specific analysis of anti-tumor ability of na?ve T cells derived from umbilical cord blood.Secondly,because autologous tumor antigen is difficult to obtain in clinical,it is hard to preparate tumor-specific DC vaccine and tumor-specific T lymphocytes,therefore,finding a clinical universal tumor antigen,is one of the current research focuses.Epithelial cell adhesion molecule(EpCAM)is one of the earliest discovered tumor-associated antigens,but it was not taken as a target for tumor therapy.EpCAM is significantly high expression in a variety of adenocarcinoma cell surface,including breast cancer,gastric cancer,colorectal cancer,pancreatic cancer and ovarian cancer.In recent years,the wide application of targeted drugs with good results has enabled the study of EpCAM as a new target entering a new stage.Studies have shown that EpCAM targeting T cells can get good clinical killing efficiency.While the research on killing efficiency using EpCAM as a targeting antigen,to activate the na?ve T cells derived from umbilical cord blood has not been confirmed in experiment.The aim of this study was to investigate the ability of expansion and activation of na?ve T cell subsets in umbilical cord blood and the anti-tumor ability in vitro and in vivo,and EpCAM-induced DC maturation,and then to prepare EpCAM-specific CTLs.Then to investigate the killing effect of CTLs to EpCAM high expressing adenocarcinoma cells in vitro and in vivo.For breaking through the individual restrictions,to achieve large-scale,industrial production of effector cells,to provide preliminary experimental data and theoretical basis.Part I Experimental study on the proliferation and activation ability of na?ve T cells derived from umbilical cord bloodObjective: Isolation and proliferation of na?ve T cells from umbilical cord blood,and verify its ability of proliferation and activation.Method: Isolation and characterization of mononuclear cells from umbilical cord blood and peripheral blood by density gradient centrifugation,CD45 RO magnetic beads were used to screen umbilical cord blood mononuclear cells,further screening of CD62L+CD45RO-T lymphocytes with CD62 L magnetic beads.MTS assay was used to detect the proliferation activity of T lymphocytes.ElISA method was used to detect the ability of T lymphocytes to secrete IFN-?.The expression of CD3,CD4,CD8,CD45 RA and CD62 L were detected by flow cytometry.Results:1 The na?ve T cells were the largest subset of umbilical cord blood and peripheral blood T cells,the proportion of na?ve T cells in umbilical cord blood is 67.57±6.53% and the proportion of na?ve T cells in peripheral blood is 50.66±5.79%.2 The proportion of na?ve T cells in umbilical cord blood was higher than that in peripheral blood(P<0.05).3 The proliferation of na?ve T cells derived from umbilical cord blood and peripheral blood was higher than that of other subsets(P<0.05).4 The proliferation ability of na?ve T cells derived from umbilical cord blood was significantly higher than that from peripheral blood(P<0.05).5 After 2 weeks of culture and expansion,the proportion of CD45 RA +CD62L + T cells in umbilical cord blood was significantly higher than that in peripheral blood(P<0.05).6 After 2 weeks of culture,the proportion of CD8+T cells increased gradually,and the na?ve T cells of umbilical cord blood was more significant(P<0.05).7 After 2 weeks of culture,compared with the na?ve T cells and other subsets of peripheral blood,the na?ve T cells from cord blood were still low in IFN-? secretion(P<0.05).Conclusion: Umbilical cord blood is rich in the na?ve T cells,and with a stronger proliferation and activation capacity relative to the origin of peripheral blood T cells.Moreover,after proliferation,the na?ve T cells derived from umbilical cord blood still with high expression of CD62 L and CD45 RA,and with low secretion capacity.Part II Experimental study of EpCAM-induced DC maturation and induction of T cell activationObjective: Mature DC cells were induced by EpCAM,and then activated na?ve T cells were used to prepare cytotoxic T lymphocyte.Method: The cells were isolated from umbilical cord blood and peripheral blood mononuclear cells by density gradient centrifugation.DC cells were obtained by adherence method.After transfection,DC cells were stimulated with cocktail(TNF-?,IL1?,IL-6,IFN-?,poly I: C)+ tumor cell lysates and cocktail+EpCAM.Flow cytometry was used to detect the expression levels of HLA-DR,CD80,CD83 and CD86 on immature DCs(imDCs)and mature DCs(mDCs).Mature DCs are co-cultured with the initial T cells,which in turn activates the initial T cells into cytotoxic T lymphocytes(CTL).The expression of CD3,CD4,CD8,CD45 RA and CD62 L on CTL cells was detected by flow cytometry.ElISA method was used to detect the ability of CTL cells to secrete IFN-?.Results:1 After the maturation of DC,the expression of HLA-DR,CD80,CD83,and CD86 on the surface of DC increased(P<0.05);2 There is no significant difference between the expression of mature DC surface antigens stimulated by cocktail+tumor lysate or cocktail+EpCAM(P>0.05);3 There was no significant difference in the expression level of umbilical cord blood derived DC and that of peripheral blood(P>0.05);4 The expression of CD45 RA and CD62 L on CTL surface was decreased,and the CTL derived from cord blood were more obvious(P<0.05).But there is no significant difference between CTL activated by different DC(P>0.05);5 The proportion of CD8+ T cells of CTL derived from umbilical cord blood was higher than those of peripheral blood derived CTL cells(P<0.05),but there was no significant difference in CTL activated by different DC(P>0.05);6 The CTL derived from umbilical cord blood have stronger IFN-?secretion than that from peripheral blood(P<0.05).The CTL cells derived from na?ve rather than memory subsets have stronger IFN-? secretion(P<0.05).There was no significant difference between CTL cells activated by different DCs(P>0.05).Conclusion: Ep CAM or tumor lysate can stimulate DC cell maturation,and can further activate the na?ve T cells become cytotoxic T lymphocytes;umbilical cord blood-derived CTL has a stronger activation and secretion capacity,in which the CTL derived from na?ve T cells have stronger activation and secretion ability.Part III The study on the specific killing effect in vitro and in vivo of CTL to EpCAM antigenObjective: To verify the specific killing effect of CTL cells in vitro and in vivo.Further evaluation of the application and prospects of umbilical cord blood na?ve T cells as a source to preparate EpCAM antigen-specific CTL.Method:1 The expression of EpCAM on the surface of LS174-T and PaCa-2 was detected by flow cytometry.2 Abbreviated name: cord blood,CB;peripheral blood,PB;na?ve T cell,n;memory T cell,m;EpCAM antigen,Ep;tumor lysate,T;cytotoxic T lymphocytes,CTL.3 The cytolysis assays of CTL derived from umbilical cord blood na?ve or memory T subset in vitro.Groups:(1)Experimental group(CTL derived from na?ve subset,CB-CTLn),including CTL induced by Ep CAM(CB-EpCTLn),and tumor lysate activated CTL(CB-T-CTLn);(2)The control group(CTL derived from memory subsets,CB-CTLm),including CTL induced by EpCAM(CB-Ep-CTLm),and CTL(CB-T-CTLm)activated by tumor lysates;(3)Blank control group;target cell: LS174-T;killing effect of effector cells on target cells by MTS method;4 The cytolysis assays of CTL derived from peripheral blood na?ve or memory T subset in vitro.Groups:(1)Experimental group(CTL derived from na?ve subset,PB-CTLn),including CTL induced by EpCAM(PB-Ep-CTLn),and tumor lysate activated CTL(PB-T-CTLn);(2)The control group(CTL derived from memory subsets,PB-CTLm),including CTL induced by Ep CAM(PB-Ep-CTLm),and CTL(PB-T-CTLm)activated by tumor lysates;(3)Blank control group;target cell: LS174-T;killing effect of effector cells on target cells by MTS method;5 The cytolysis assays of CTL derived from cord blood or peripheral blood na?ve T subset in vitro.Group:(1)The experimental group(CB-Ep-CTLn,CB-T-CTLn);(2)The control group(PB-Ep-CTLn,PB-T-CTLn);(3)Bl-ank control group(PBS).6 To test the specific killing effects of CTLs in vitro.Groups:(1)The experimental group(CB-Ep-CTLn,CB-Ep-CTLm,PB-Ep-CTLn,PB-EpCTLm),Target cells: LS174-T;(2)The control group(CB-Ep-CTLn,CB-EpCTLm,PB-Ep-CTLn,PB-Ep-CTLm),Target cells: PaCa-2,LS174-T(Blocking EpCAM antigen on cell surface with anti-EpCAM antibody);(3)Blank control group(PBS).Target cells::LS174-T,PaCa-2;The killing effect of effector cells on target cells was detected by MTS.7 Animal models: Four weeks old,female,NOD/SCID mice were usedfor in vivo studies.Experimental animals: 4 weeks old NOD/SCID mice.1 ×106 target cells(0.1ml)were injected subcutaneously in the left forelimb of the mice.After 10 days,the mean tumor size was 0.5mm3(volume=length×width2÷2).8 In vivo antitumor effect of CTL prepared from cord blood na?ve T cells and memory T cells.Group:(1)The experimental group(CB-Ep-CTLn,CB-TCTLn);(2)The control group(CB-Ep-CTLm,CB-T-CTLm);(3)Blank control group(PBS);LS174-T tumor-bearing mice were injected with 1×107 effector cells in the tail vein,and the tumor size was measured every other day.The tumor volume reached 2mm3 as the observation end point.9 In vivo antitumor effect of CTL prepared from peripheral blood na?ve T cells and memory T cells.Group:(1)The experimental group(PB-Ep-CTLn,PB-T-CTLn);(2)The control group(PB-Ep-CTLm,PB-T-CTLm);(3)Blank control group(PBS);LS174-T tumor-bearing mice were injected with 1×107effector cells in the tail vein,and the tumor size was measured every other day.The tumor volume reached 2mm3 as the observation end point.10 In vivo specific antitumor effect validation.Group:(1)The experimental group(CB-Ep-CTLn,CB-Ep-CTLm,PB-Ep-CTLn,PB-Ep-CTLm),experimental animals: LS174-T tumor-bearing mice;(2)The control group(CB-Ep-CTLn,CB-Ep-CTLm,PB-Ep-CTLn,PB-Ep-CTLm),experimental animals: PaCa-2 tumor-bearing mice;(3)Blank control group(PBS);experimental animals: LS174-T tumor-bearing mice,PaCa-2tumor-bearing mice were injected with 1×107 effector cells in the tail vein,and the tumor size was measured every other day.The tumor volume reached2mm3 as the observation end point.Results:1 The expression of Ep CAM was 99.22±0.42% on LS174-T cell and5.69±0.89% on PaCa-2 cell surface.2 CB-Ep-CTLn had a stronger cytotoxic effect on LS174-T cells than CB-Ep-CTLm(P<0.05).CB-T-CTLn was more effective than CB-T-CTLm on LS174-T cells in vitro cytotoxic effect(P<0.05).CB-Ep-CTLnhas a strongercytotoxic effect on LS174-T cells than CB-T-CTLn(P<0.05).CB-Ep-CTLm and CB-T-CTLm had no significant difference in the in vitro killing of LS174-T cells(P>0.05).3 PB-T-CTLn had a stronger cytotoxic effect on LS174-T cells than PB-Ep-CTLm(P < 0.05),and PB-T-CTLn had more specificity than PB-T-CTLm on LS174-T cells.PB-Ep-CTLn had a stronger cytotoxic effect on LS174-T cells than PB-T-CTLn(P<0.05).There was no significant difference in the in vitro killing of LS174-T cells between PB-Ep-CTLm and PB-T-CTLm(P>0.05).4 CB-Ep-CTLn had a stronger cytotoxic effect on LS174-T cells than PB-Ep-CTLn(P < 0.05).There was a significant difference between CB-T-CTLn and PB-T-CTLn in vitro cytotoxic effect on LS 174-T cells(P<0.05).5 CB-Ep-CTLn and PB-Ep-CTLn have strong killing effect in vitro of LS174-T,have a very weak cytotoxic effect on PaCa-2 in vitro(P<0.05);anti-EpCAM antibody has been added in the target cells of LS174-T,the killing effect of CB-Ep-CTLn and PB-Ep-CTLn on LS174-T target cells was decreased with statistical significance(P<0.05).6 CB-Ep-CTLn had a stronger antitumor effect on LS174-T tumorbearing mice than CB-Ep-CTLm(P < 0.05).CB-T-CTLn had a stronger antitumor effect on LS174-T tumor-bearing mice than CB-T-CTLm(P<0.05),and CB-Ep-CTLn was more effective than CB-T-CTLn on LS174-T tumor-bearing mice(P<0.05).7 PB-Ep-CTLn had a stronger antitumor effect on LS174-T tumorbearing mice than PB-Ep-CTLm(P < 0.05).PB-T-CTLnhad stronger antitumor effect on LS174-T tumor-bearing mice than that of PB-T-CTLm(P< 0.05).PB-Ep-CTLn had a stronger antitumor effect on LS174-T tumor-bearing mice than PB-T-CTLn(P<0.05).8 CB-Ep-CTLn had a stronger antitumor effect on LS174-T tumor-bearing mice than PB-Ep-CTLn(P<0.05).CB-T-CTLn had a stronger antitumor effect on LS174-T tumor-bearing mice than PB-T-CTLn(P<0.05).9 CB-Ep-CTL had a strong antitumor effect on LS174-T tumor-bearing mice and had no significant effect on PaCa-2 tumor-bearing mice(P<0.05).PB-Ep-CTL had stronger effect on LS174-T tumor-bearing mice and the antitumor effect on PaCa-2 tumor-bearing mice was not obvious(P<0.05).Conclusion:1 CTLs prepared from the na?ve T cells derived from cord blood or peripheral blood have stronger anti-tumor ability than other subgroups in vitro and in vivo.2 CTLs induced by EpCAM has stronger anti-tumor ability than CTLs induced by tumor lysates.3 CTLs prepared from cord blood na?ve T cells have a stronger anti-tumor ability than CTLs prepared from peripheral blood na?ve T cells.4 CTLs induced by EpCAM has a specific killing ability against high EpCAM-expressing tumors.