| Malignant neoplasm remains one of the leading causes of death worldwide.For decades,the treatment of malignant tumors has challenged the survival of cancer patients because of the lack of reliable and effective treatment methods.Postoperative recurrent metastatic or unresectable advanced tumors must be treated by traditional means such as radiotherapy and chemotherapy,but the clinical efficacy is often limited and accompanied by severe adverse reactions.The progress and development of tumor immunotherapy make the use of immunotherapy to treat cancer more and more important.The researchers have developed a variety of strategies to activate the immune response.Among them,IL-2 is one of the earliest immune-enhancing drugs,because it can promote the proliferation and differentiation of T cells and enhance the function of T cells,and has been approved for the treatment of renal cell carcinoma,melanoma and non-hodgkin lymphoma,and has become one of the earliest methods of tumor immunotherapy.However,the limitations of first-generation immunotherapy lie in its low response rate and high incidence of adverse events.Find reliable immunotherapeutic strategies and methods to enhance T by regulating cell activation and proliferation.The therapeutic effect becomes the main research direction of tumor immunotherapy.Recent studies have found that immune detection sites play an important regulatory function in the process of T cell activation and killing.Among them,immunosuppressive molecules such as cytotoxicity T lymphocyte antigen 4(Cytotoxic T-Lymphocyte Antigen 4,CTLA-4),programmed death receptor-1(programmed cell death-1,PD-1),IDO-1,Tim3 have been paid more and more attention to the immune regulation function of T cells.Studies have shown that the expression of CTLA-4 in the early stages of T cell activation increases,the ligand exerts competitive inhibition B7 binding,weakening the B7-CD28 costimulatory signaling pathway,which leads to the "immune brake" effect and hinders the activation of T cells.T cell activation is accompanied by a large amount of INF-? release,resulting in a large increase in the PD-1 on the surface of T cells,and a "T depletion" caused by the binding of its ligand,which leads to the decrease of cell activation,proliferation and killing function.thus,recent studies on programmed death factor 1(PD-1)and cytotoxic T lymphocyte antigen 4(CTLA-4)have progressed rapidly.The research and treatment of immune checkpoints is the focus of current immunotherapy research[1].the discovery of two T cell co-inhibiting molecular CTLA-4 and PD-1 makes cancer immunotherapy and targeted therapies revolutionize how to treat cancer,especially in patients with advanced disease [2].Therefore,a large number of preclinical and clinical studies are carried out around PD-1 and CTLA-4,in which immunotherapy targeting PD-1 has more in-depth research in anti-tumor,anti-infection,anti-autoimmune diseases and organ transplantation.Immunotherapy targeting CTLA-4,anti-tumor immunity,etc.aspects also showed significant therapeutic effects.With the in-depth study of CTLA-4 and PD-1 blockers and their success in cancer therapy,targeted PD-1 and CTLA-4 antibody drugs are rapidly emerging and approved for marketing,and are gradually applied to clinical treatment of multiple malignant solid tumors[3].CTLA-4 is one of the immune checkpoints successfully applied to the clinic.Monoclonal antibody targeting drugs(ipilimumab)targeting CTLA-4 have been approved for the treatment of diseases such as melanoma,and follow-up studies have shown to be equally effective in the treatment of multiple tumors such as lung cancer.7 targeted CTLA have been approved to CTLA-4/PD-1 drugs for the treatment of various types of cancer.T lymphocytes are known to be the main immune cells in the human body.Among them,CD4~+T cells are mainly expressed in helper T cells(Th),which are called helper T cells(helper T cell),while CD8 T cells are called cytotoxic cells(cytotoxic T cell,CTL)that specifically kill target cells by means of cell lysis and apoptosis,which are associated with cellular immunity and antitumor effects.The primary function of CD4~+T cells is to assist CD8~+Tcells in eliminating viruses,bacteria,and abnormalities in the body raw tumor cells.Numerous studies have shown that PD-1 blockers enhance CD4~+T and CD8~+T cellular responses.The aim of this study was to determine whether blocking CTLA-4 immunosuppressive molecules with antibodies could effectively enhance cytotoxicity CD8~+T the antitumor effect of lymphocytes.To this end,we examined whether PD-1 and CTLA-4 were highly expressed on activated T cells by studying the characteristics of cell PD-1 and CTLA-4 expression in peripheral blood of bladder cancer patients.Subsequently,the immune negative regulatory molecules on the surface of peripheral blood T cells were blocked by antibody CTLA-4,and whether the antibody could enhance the cellular immune response and cytotoxicity was tested to verify the blocking of cytotoxic T lymphocyte surface expression of CTLA-4 molecules,and(cytotoxic T cell,CTL)whether it can enhance its killing efficiency in vitro and the anti-tumor effect of subcutaneous transplanted tumor model.Part one Experimental Study on PD-1 and CTLA-4 Expression Characteristics of Immunosuppressive MoleculesObjective: To detect the expression characteristics of PD-1 and CTLA-4 co-inhibitory molecules in peripheral blood monocytes of bladder cancer patients and healthy volunteers.Method: 1.Patients with bladder cancer(n=62)and healthy volunteers(n=32)received 50 ml,heparin anticoagulant and diluted 1 times PBS.absorbent white film layer(PBMC)was obtained by centrifugation by Ficoll method and placed in a 50 ml centrifuge tube.the PBS,800×g?10min was centrifuged and washed twice.Get PBMC.after washing platelets Through isolation culture,adherent cells cultivate DC cells,detect DC phenotypes,and suspension cells cultivate CIK cells.2.CD3 antibodies stimulate T cell activation.to detect the ability of T lymphocytes to secrete IFN-? ? TNF-? in peripheral blood.T lymphocyte specific expression CD3,and further divided into CD4~+T cells,CD8~+T cell subsets;3.The expression characteristics of PD-1 and CTLA-4 in CD8~+T and CD4~+T cells of bladder cancer patients and healthy volunteers were measured by flow cytometry;4.The correlation between PD-1 and CTLA-4 expression and the age,sex,histological grade,tumor size and other clinical parameters of bladder cancer patients was analyzed.Results: 1.There was no statistical difference in the expression of PD-1 in CD4 T cells in bladder cancer patients compared with healthy volunteers(mean 14.7%±3.5% vs 13.6%,4.6%,Fig.1A).in contrast,PD-1 expression in CD8 T cells was significantly higher than in healthy volunteers(mean frequency 12.8%±4.5% vs 8.1%,2.9%,Fig.1B).2.The expression frequency of CTLA-4 in CD4 T cells was not statistically different compared with bladder cancer patients and healthy blood donors(Fig.1C).Meanwhile,in patients with bladder cancer,the expression of CTLA-4 in CD8~+T cells was higher than that in healthy controls(mean 10.2%±3.2% vs 7.5%±2.8%)(Fig.1D).3.There was no significant correlation between PD-1 expression on CD8 T cells and any clinical parameters(table 1).Nevertheless,there was a close correlation between CTLA-4 expression and tumor size and TNM staging(table 1).The comparison is statistically significant.Conclusion: The expression of PD-1 and CTLA-4 in peripheral blood of bladder cancer patients was significantly higher than that of healthy volunteers.Part Two Study on Anti-tumor Effect of Blocked CTLA-4 CD8~+T Lymphocytes in VitroObjective: The in vitro killing effect of DC-CTL after blocking antiCTLA-4 antibody on tumor cells was detected.Method: 1.The peripheral blood of tumor patients was collected,the mononuclear cells were extracted Focill the method,then the DC cells and T cells were isolated by adherent method.2.The expression level of CTLA-4 ligand CD80/86 in five bladder cancer cells was detected,and CD80/86 highly expressed cell lines were selected as target cells.3.T cell culture was expanded to 7 days,the CTLA-4 was sealed with anti CTLA-4 antibody and then prepared with DC vaccine.The expression of CTLA-4 was detected by flow cytometry.4.The as-prepared tumor-specific cytotoxic T cells(CTL)were grouped: group 1: non-intervention group CTLs(specific cytotoxicity T lymphocytes);group 2: saline blank control group;group 3: anti-antibody treatment group,the cell proliferation of the CTL was detected every 7 days for a duration of 21 days;and the expression level of the CTLA-4 in the three groups was detected by flow cytometry.5.ELISA assay for the amount of IFN-? and TNF-? secreted in the medium of the experimental group CTL and the control group CTL;6.The experimental group and the control group were tested with 96-well plate cells in vitro.Results: 1.The expression of CTLA-4 CTLA-4 was detected by flow cytometry after the closure of anti CTLA-4 antibody.the expression of CTLA-4 in the anti CTLA-4 treatment group was significantly inhibited compared with that in the control group(P<0.05)(figure).1A).the expression of CTLA-4 on CTL in the control group increased from 9.1% to 17.6%;in the CTLA-4 blocking group,the expression of CTLA-4 increased from 4.1% to 9.2%.2.The cell proliferation CTL the unantibody intervention group CTL?the blank control group and the anti CTLA-4 treatment group was tested every 7 days for 21 days.we found that CTL proliferation was not affected by CTLA-4 blockade during culture.With the prolongation of time,the proliferation of cells gradually increased,and there was no significant difference in cell proliferation in different groups(Fig.1B).3.The change of cytokine IFN-? and TNF-? secretion ability was detected by ELISA assay in the supernatant CTL the anti-drug treatment and control groups.The IFN-? and TNF-? levels produced by the CTL after CTLA-4 blockade were higher(Fig.1C).4.CTLA-4 ligand CD80/86 expression levels were detected in 5 bladder cancer cell UM-UC-3?TCCSUP?J82?T24?5637,with the highest T24 expression and the lowest UM-UC-3 expression levels,which were statistically significant compared with different tumor cells(P>0.05)(fig.1D).5.The cytotoxicity of anti-CTLA-4 antibody-blocking CTLs to T24 cells was significantly increased and dose-dependent.CTLA-4 antibody blocking on the CTL enhanced the anti-tumor activity of the cells.Conclusion: CTLA-4 induced T cell inactivation is considered to be a mechanism of immunosuppression CTL antitumor activity against bladder cancer cells after blocking the CTLA-4/B7 pathway.Part three The Experimental Study anti-tumor effect of blocking CTLA-4 CD8~+T lymphocytes in vivoObjective: To further verify the CTL anti-tumor immune killing effect after anti-CTLA-4 closure,and to evaluate the specific in vivo anti-tumor effect after CTL closure.Method: 1.Animal model building: human-derived mononuclear cells were used to establish SCID model mice with human immune function,and xen model of T24 cells was established,and the efficiency of flow detection and reconstruction was established.immune reconstituted mice 5-week-old male SCID mice(chinese academy of sciences)were injected intravenously with 4×107 peripheral blood lymphocytes with a tumor diameter of 6-9 mm,to establish a SCID mouse model.2.Preventive testing The 1×107 CTLs after anti-CTLA-4 antibody blockade and the control group were injected through the caudal vein and then subcutaneously transplanted T24 cells in mice at a dose of 1×106 cells suspended in 100?l PBS(n 5 per group)(figure 1 A).tumor volume(n=5)was measured in mice after the anti-CTLA-4 antibody blocking group or control CTL from the beginning of inoculation(figure 1 B).PCNA immunohistochemical staining of tumor tissues was performed using anti-CTLA-4 antibody blockade or control group CTL(figure 1 C).3.For therapeutic assay,T24 cells were first inoculated in SCID mice.when the tumor grew to close to 100 mm 3,mice were randomly divided into groups,and 1×107 experimental groups control groups treated with antiCTLA-4 antibodies were injected intravenously(n=5,each group).Feeding conditions follow animal center guidelines: temperature 20-22?;humidity 50-70%;normal diet,free of specific pathogens.tumor volume was measured with vernier caliper every 3 days.The mice were killed 27 days later.monitoring tumor load by calculating tumor volume.tumor volume is calculated as follows: volume =(length)×(width)2/2.tumor volume(n =5)was measured in mice CTL the anti-CTLA-4 antibody blocking group or control group since the beginning of inoculation(fig1E).PCNA immunohistochemical staining of tumor tissues was performed using anti-CTLA-4 antibody blockade or control group CTL(fig 1 F).killing experiments of CTL cells prepared after CTLA-4 antibody closure.4.Group 1)CTLA-4 antibody blocked CTLs experimental group;2)control group;3)saline control group(NS group)5.Statistical analysis Data were reported as mean ± standard deviation from at least three independent experiments.Statistical analysis was performed using SPSS(v13.0,chicago,il,usa).The correlation between CTLA-4 expression and clinical parameters was compared by ?2 test.Student's t-tests were used to assess intergroup differences in in vitro and in vivo assays.ANOVA was used to compare between multiple groups.P<0.05 was statistically significant.Results: 1.The humanized SCID mouse model was constructed by the tail vein injection monocyte method.the humanized transplanted tumor model mice were divided into: CTLA-4 antibody blocking group and CTL control group.compared with the control group,the tumor growth in the anti CTLA-4 antibody blocking group was significantly slowed down,which indicated that the tumor growth was delayed after being blocked by anti-antibody.Meanwhile,the PCNA positive cells in the CTLA-4 antibody test group also decreased significantly.The cell proliferation CTL the unantibody intervention group CTL?the blank control group and the anti CTLA-4 treatment group was tested every 7 days for 21 days.we found that CTL proliferation was not affected by CTLA-4 blockade during culture.With the prolongation of time,the proliferation of cells gradually increased,and there was no significant difference in cell proliferation in different groups(Fig.1B).2.Prophylactic test: 1×107 doses of control CTL and anti-CTLA-4 antibody-blocked CTL-treated cells,then subcutaneously transplanted T24 cells at a dose of 1×106 cells(Fig.1A,each group(n = 5)Compared with the control group,the tumor growth of the anti-CTLA-4 CTLs experimental group was significantly slower,indicating that the anti-CTLA-4 CTLs delayed the latency of T24 xenografts(as shown in Figure 1B),and at the same time,anti-CTLA-4 PCNA positive cells in the CTL group were also significantly reduced(Fig.1C).3.Therapeutic experiments: Compared with the control group,the tumor volume of the anti-CTLA-4 CTLs treatment group was significantly reduced,indicating that the anti-tumor activity of anti-CTLA-4 CTLs was enhanced in vivo(n=5 per group)(Fig.1E).Similarly,the anti-CTLA-4 antibody-blocked CTLs also showed a decrease in PCNA and immunoreactivity in the CTLs(Fig.1F).4.The anti-CTLA-4 antibody-blocked DC-CTL treatment group had stronger anti-tumor killing effect on T24 cells than the DC-CTL treatment group;the anti-CTLA-4 antibody-blocked DC-CTL treatment group was more physiologically controlled than the saline control group(The NS group had a stronger anti-tumor effect on T24 cells,which was statistically significant.The DC-CTL treatment group had stronger anti-tumor killing effect than the saline control group(NS group),and the two were statistically significant.5.ELISA assay was performed to detect cytokine IFN-? and TNF-? secretion in the supernatant: after antibody blockade produced higher levels of IFN-? and TNF-?(table 1).Conclusion: 1.CTL enhances its immune response and cytotoxicity to bladder cancer cells after anti-CTLA-4 antibody is blocked.2.CTL showed better anti-tumor activity in a subcutaneous xenografted SCID mouse model after anti-CTLA-4 antibody blocking.3.CTLs after anti-CTLA-4 antibody blockade produce higher levels of IFN-? and TNF-?.4.Tumor growth was significantly slowed in the anti-CTLA-4 CTL group,indicating that anti-CTLA-4 CTL delayed tumor growth. |
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