| With the advances of science and technology,people are constantly exploring,innovating,and seeking more effective treatment methods to malignant tumors.Nowadays,cancer immunotherapy came into being,which regulate the tumor microenvironment,enhance the body's anti-tumor immune response,and finally control tumor progression and even eliminate tumor.The tumor microenvironment is composed of stromal cells(fibroblasts,endothelial cells,macrophages,lymphocytes,etc.),extracellular matrix(ECM),and a series of soluble factors.Tumor cells interact with the tumor microenvironment and then lead to tumorigenesis and tumor progression.Myeloid-derived suppressor cells(MDSCs)and regulatory T cells(Tregs)are important immunosuppressive cells which directly involved in tumor progression in the tumor microenvironment.Cancer immunotherapy includes checkpoint blockade immunotherapy,tumor vaccine,adoptive cell therapy,etc.Among them,checkpoint blockade immunotherapy is targeted the T cell inhibitory checkpoint proteins such as CTLA-4 and PD-1,restores the immune activity of T cells,and then restores the body's immune surveillance,and maintains immune homeostasis.CTLA-4 is mainly induced by T lymphocytes activation and is highly homologous in structure to CD28.CTLA-4 inhibits T cells activation signal transduction by competitively binding B7 ligands(CD80 and CD86)on antigen presenting cells.CTLA-4 is highly expressed in tumor-infiltrating Tregs.With the treatment of anti-CTLA-4 mAb(monoclonal antibody),macrophages can selectively deplete the tumor-infiltrating Tregs through antibody-dependent cell-mediated cytotoxicity(ADCC)in the tumor microenvironment,and the effector function of CD4+Thl cells have great enhancement,and finally exert anti-tumor effects.PD-1 is mainly expressed on the surface of activated T lymphocytes,B lymphocytes,monocytes and natural killer(NK)cells.By blocking the signaling pathway of PD-1/PD-L1(the ligand of PD-1),the effector function of exhausted-like tumor-infiltrating CD8+T cells is reversed,thereby restoring the body's anti-tumor immune response.Many preclinical trials and clinical trials have shown that checkpoint blockade immunotherapy is widely used in the treatment of a variety of advanced tumors,including melanoma,colorectal cancer,non-small cell lung cancer,lymphoma.This immunotherapy effectively inhibits tumor growth and metastasis,and prolongs progression-free survival of patients.However,the response rate of immune checkpoint inhibitors is still limited,and many patients are nonresponsive or resistant to treatment,which also suggests that checkpoint blockade immunotherapy combined with traditional/new-type treatments such as chemotherapy,radiation,epigenetic modulators,small molecule targeted therapies,or other therapies may be a good choice to increase the systemic anti-tumor immune response and reduce immune-related adverse events(irAE).The cytokine IL-33 is a member of the IL-1 superfamily.It is constitutively expressed in the nucleus of epithelial cells,endothelial cells and fibroblast cells,and plays an important role in innate and adaptive immunity by binding to its receptor ST2.Studies have shown that IL-33 can participate in both Th2 type inflammatory immune response and Thl type anti-tumor immune response.The IL-33/ST2 pathway can increase the number of tumor-infiltrating CD8+T cells and NK cells,and promote the secretion of effector molecules such as IFN-y and perforin,then inhibit tumor growth and metastasis in mouse models of B16 melanoma and Lewis lung cancer.The infiltration and function of T cells in the tumor microenvironment determine the outcome of the body's immune response to tumor.Although terminally differentiated effector T cells play an anti-tumor effector function,they are difficult to survive for a long time.Memory T cells are essential for maintaining long-term immune memory and anti-tumor immune response.Infiltration of memory T cells in the tumor microenvironment is one of the important indicators for assessing the prognosis of patients.Memory T cells can be divided into central memory T cells(CD44+CD62L+TCM),effector memory T cells(CD44+CD62L-TEM)and tissue-resident memory T cells(CD103+TRM).They facilitate immune surveillance and better control of persistent infections during secondary and/or chronic infections.Studies have shown that the number of tumor infiltrating CD103+CD8+T cells is significantly positively correlated with the good prognosis of patients with lung cancer,ovarian cancer,melanoma and other tumors.TGF-?,TNF-? and IL-33 can up-regulate the expression of CD103 on CD8+T cells.IL-33 promotes the proliferation of CD4+Foxp3+Tregs to a certain extent.Several studies have indicated that CTLA-4 is constitutively expressed on regulatory T cells.Therefore,this study aims to reveal the significant role of the IL-33/ST2 pathway in the checkpoint blockade immunotherapy by anti-CTLA-4 and anti-PD-1 antibody,and establish a new cancer immunotherapy model of IL-33 combined with immune checkpoint blocking antibodies to provide new ideas for clinical cancer immunotherapy.Part I:Research on the mechanism of cytokine IL-33 combined with immune checkpoint blocking antibody to CTLA-4 in the treatment of malignant tumors[Obj ective]In a mouse melanoma tumor-bearing model,to investigate the effects and possible mechanisms of cytokine IL-33 combined with immune checkpoint blocking antibody of anti-CTLA-4 in the treatment of malignant tumors.[Methods]1.WT mice were randomly divided into groups and subcutaneously inoculated with mouse melanoma cells B16 or B16-IL-33,2×105 cells/50?l/mouse on the right side of the abdomen to establish melanoma mouse models.When tumors are visible on day 6 after tumor inoculation,the treatment of immune checkpoint blocking antibodies was started.The IgG control antibody or anti-mouse CTLA-4 antibody at a dose of 200?g/100?l/time are injected intraperitoneally.The treatment was performed every 4 days for a total of 4 times.At the same time,the tumor size and survival of mice were recorded every 2 days from the day of tumor inoculation,and the tumor growth curve and mouse survival curve were plotted.2.On the 10th day(the day after the second antibody treatment)and the 18th day(the day after the fourth antibody treatment),tumors form four groups of mice were collected.Real-time PCR was used to analyze the expression of IFN-?,TNF-?,and other effector molecules in tumor microenvironment at different time points of treatment.3.On the 11th day(the second day after the second antibody treatment)and the 19th day(the second day after the fourth antibody treatment),tumors from four groups of mice were collected,labeled by immunofluorescence and flow cytometry.The infiltration of immune cell populations and their effector functions in tumor microenvironment at different timepoints of treatment were analyzed at the cell level.[Results]1.Cytokine IL-33 combined with anti-CTLA-4 mAb treatment significantlyinhibited the growth of melanoma and prolonged the survival time of tumor-bearing mice,the difference was statistically significant,P<0.05;2.Flow cytometry was used to detect the infiltration of each immune cell populationsin the tumor microenvironment.The infiltration of CD45+cells and CD8+T lymphocytes increased,and the infiltration of CD4+T lymphocytes increased much more significantly in the combination group,P<0.05;3.On the 19th day,the IFN-? secretion of CD4+T lymphocytes and CD8+T lymphocytesin the tumor microenvironment increased in the treatment groups,and the combination group increased most significantly;4.On the 11th day and the 19th day,Foxp3+CD4+Tregs decreased in the immunecheckpoint blocking antibody treatment groups,but IL-33 tended to increase Foxp3+CD4+Tregs;at the same time,the ratio of IFN-?+effector T cells to Foxp3+CD4+Tregs(Teffs/Tregs)in CD4+T lymphocytes and CD8+T lymphocytes have an increasing trend in the immune checkpoint blocking antibody treatment groups;5.On the 19th day,Gr-1hiCD11b+MDSCs in the tumor microenvironment were significantly reduced in the treatment groups,and the reduction was the most significant in the combination group,P<0.05;6.IL-33 increased the infiltration of Tcm and TRM cells in CD4+T lymphocytes and CD8+T lymphocytes,and anti-CTLA-4 antibody treatment groups increased the infiltration of TEM cells in CD4+T lymphocytes and CD8+T lymphocytes;7.On the 10th day and the 18th day,Real-time PCR results showed that,compared with the control group,GZM-B,TNF-?,and IL-12 were highly expressed in the combination group,P<0.05;8.On the 3 0th day,in the two groups inoculated with BI6-IL-3 3 tumors,compared with the control group,the expression of IFN-y,perforin,and TNF-? in the tumor microenvironment of the combination group were up-regulated;and CD 103 and CD69 expressions in the combination group were also significantly up-regulated,P<0.05.[Conclusion]Cytokine IL-33 combined with immune checkpoint CTLA-4 blocking antibody can increase the infiltration of immune cell populations in the tumor microenvironment,including CD45+cells,CD4'T lymphocytes,and CD8+T lymphocytes,and promote effector molecules secretion;it inhibits the expression of Tregs and MDSCs in the tumor microenvironment;at the same time,it up-regulates the expression of memory T cells,especially CD103+TRM cells,thereby enhancing the body's anti-tumor immune response,effectively suppressing tumor growth and delaying survival of tumor-bearing mice.In the end,it provides experimental basis and clinical application possibilities for a new therapeutic model of cytokine IL-33 combined with immune checkpoint blocking antibody.Part ?:Research on the mechanism of cytokine IL-33 combined with immune checkpoint blocking antibodies to PD-1 and CTLA-4 in the treatment of malignant tumorsPrevious research by our lab showed that in Humanized PD-1 KI/KO mice model,IL-33 combined with immune checkpoint PD-1 blocking antibody can enhance the infiltration and effector function of immune cells in the tumor microenvironment,promote the anti-tumor immune response significantly,thus tumor growth in tumor-bearing mice is significantly suppressed and the survival time is prolonged.Therefore,this article combines the cytokine IL-33 with the checkpoint molecules PD-1 and CTLA-4 blocking immunotherapy to observe whether the three can further enhance the anti-tumor immune response in synergy,and to provide corresponding theoretical basis for clinical cancer immunotherapy.[Objective]In a mouse melanoma tumor-bearing model,to investigate the effects and possible mechanisms of cytokine IL-33 combined with immune checkpoint blocking antibody of anti-PD-1 and anti-CTLA-4 in the treatment of malignant tumors.[Methods]1.Humanized PD-1 KI/KO mice were randomly divided into groups and subcutaneously inoculated with mouse melanoma cells B16 or B16-IL-33,2×105 cells/50?l/mouse to establish a melanoma mouse model.Antibody treatment is implemented when the tumors are visible from the inoculation site.Injected intraperitoneally with PBS(to avoid the interference with IgG antibody),Humanized anti-PD-1 mAb,Anti-mouse CTLA-4 antibody,and Humanized anti-PD-1 mAb+Anti-mouse CTLA-4 antibody,at a dose of 200?g/100?l/time,treated once every 4 days,for a total of 4 times.At the same time,the tumor size and survival of mice were recorded every 2 days from the day of tumor inoculation,and the tumor growth curve and mouse survival curve were plotted.2.On the 10th day(the day after the second antibody treatment)and the 18th day(the day after the fourth antibody treatment),eight groups of mouse tumors were collected.Real-time PCR was used to analyze the expression of effector molecules in tumor microenvironment at different time points of treatment.3.On the 11th day(the second day after the second antibody treatment)and the 19th day(the second day after the fourth antibody treatment),tumors from eight groups of mice were collected,labeled by immunofluorescence and flow cytometry.The infiltration of immune cell populations and their effector functions in tumor microenvironment at different time points of treatment were analyzed at the cell level.[Results]1.Cytokine IL-33 combined with immune checkpoint PD-1 and CTLA-4 blocking antibodies(the triple combination treatment group)can significantly inhibited the growth of melanoma and prolonged the survival time of tumor-bearing mice,the difference was statistically significant,P<0.05;2.Flow cytometry was used to detect the infiltration of each immune cell population in the tumor microenvironment.The triple combination treatment group significantly increased the infiltration of CD45+cells and CD4+T lymphocytes,P<0.05;and CD8+T lymphocyte infiltration also tends to increase;3.In the tumor microenvironment,IL-33 increased the infiltration of Tcm and TRM cells in CD4+T lymphocytes and CD8+T lymphocytes,P<0.05;and immune checkpoint PD-1 and CTLA-4 blocking antibodies treatment groups increased the infiltration of TEM cells in CD4+T lymphocytes and CD8+T lymphocytes;4.Real-time PCR results showed that,on the 10th day and the 18th day,compared with the control group,CD 103 and CD69 were highly expressed in the triple combination treatment group,P<0.05.[Conclusion]The combination of cytokine IL-33 and immune checkpoint PD-1 and CTLA-4 blocking antibodies can further increase the infiltration of immune cell populations in the tumor microenvironment,including CD45+cells,CD4+T lymphocytes,and CD8+T lymphocytes;at the same time,further up-regulate the expression of TRM cells,thereby continuously enhance the body's anti-tumor immune response,maximally inhibit tumor growth and delay tumor-bearing mice's survival time. |
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