| Eomesodermin(Eomes)is a member of the transcription factor family containing T-Box Domain and plays an important role in the generation and regulation of effector T cells and memory T cells.Memory T cells are mainly divided into central memory T cells(TCM)and effector memory T cells(TEM)which can be classified by their localization,as Tcm are more easily circulated to peripheral lymphoid tissues and TEM are stimulated to rapidly differentiate into effector T cells.Recently,a group of CD8+ memory T cell subsets,called tissue resident memory T cells(TRM),has been discovered as an important effector cell group in tissue regional immunity.High level of Eomes expression inhibits the formation of TRM cells in the skin and lung,and down regulation of Eomes is critical for the differentiation of TRM cells.Integrin CD103 is a classic surface marker of TRM cells and is an important homing receptor that leads to the migration of leukocytes in specific peripheral tissues.CD 103 mediates cell adhesion,migration,and signaling through interaction with its ligand E-cadherin.Moreover,CD103 directly promotes the release of perforin and granzymes by CD8+CTL cells,thereby enhancing the anti-tumor immune response.The abundant transforming growth factor-(3(TGF-P)in the tumor microenvironment can induce the expression of CD103 on the surface of CD8+T lymphocytes.Inhibition of TGF-β expression by neutralizing antibodies or inactivating the CD8 receptor reduces the number of CD103+CD8+T cells in the tissue.CD103+CD8+TRM cells infiltration into tumor tissues is associated with the prognosis of various cancers.CD4+Th9 cell-derived IL-9 has anti tumor properties in solid tumors such as lung adenocarcinoma and melanoma.The plasticity of Tc9 cells enables them to differentiate into Tcl-like effector cells that produce long-lasting IFN-y.Utilizing Th9 and Tc9 cells provide new strategies and approaches to improve adoptive immunotherapy.In view of this,we investigated the function of Eomes in IL-33-mediated tumor immune response and its possible mechanisms by means of Eomes deficient mice,and further explored the role of Eomes in IL-33 regulation of the effector function of mouse CD4+Th9 and CD8 Tc9 cells cultured in vitro.In order to provide a theoretical basis and new ideas for the potential application of CD4+Th9 and CD8+Tc9 cells in adoptive immunotherapy.Part I The role and mechanism of Eomes in IL-33-mediated tumor immune response[Objective]To explore the role of Eomes gene in IL-33-mediated tumor immune response and its possible mechanisms.[Methods]Using WT mice as control,B16 and B16-IL-33 melanoma tumor-bearing mouse models were constructed by using Eomes KO mice.Tumor sizes were recorded to assess the tumor growth curves.Survival rate of the mice was observed,providing the survival curve.On the 18th day after tumor inoculation,the infiltration of immune cell population and its effect function in tumor microenvironment were detected by flow cytometry.On the 11th,17th and 24th day of tumor growth,Real-time PCR was used to detect changes in mRNA levels of various molecules in tumor tissues.[Results]1、Compared with WT mice,there was no significant difference in B16 tumor growth in Eomes KO mice,but B16-IL-33 tumors were significantly inhibited in Eomes KO mice with statistically significant difference(P<0.05).The survival time of mice was prolonged.A significant difference was also seen in survival curves(P<0.05).2、Deficient of Eomes gene enhanced the expression of IFN-y,perforin and Granzyme B mRNA in IL-33-mediated tumor microenvironment,the difference was statistically significant(P<0.05).3、Deficient of Eomes gene promoted the expression of IL-12 and IL-15 mRNA in IL-33-mediated tumor microenvironment,which was statistically significant(P<0.05).4、Deficient of Eomes gene increased the infiltration of CD103+CD8+TRM cells in the tumor microenvironment of mice,and the difference was observed in a statistical significance(P<0.05),but had no effect on CD69 expression.5、Deficient of Eomes gene up-regulated the expression of IL-9 and TGF-β mRNA in IL-33-mediated tumor microenvironment,that was found to be statistically significant(P<0.05).6、On the 29th day after inoculation of B16-IL-33 cells,B16-IL-33 tumors were re-inoculated on the opposite side of that individual mouse,and the tumor growth in the EKO group was significantly slower than that in the WT group,and finally the tumor was completely disappeared.[Conclusion]Deficient of Eomes gene enhanced IL-33-mediated anti-tumor effect,significantly inhibited tumor growth in mice models and prolonged survival time of tumor-bearing mice.Eomes KO mice were observed with enhanced IL-33-mediated proliferation of effector cells in host anti-tumor capacity,effector function,and memory cell response,possibly by promoting IL-33-mediated infiltration of CD103+CD8+TRM cell subsets in the tumor microenvironment,or up-regulating IL-33-mediated expression of IL-9,which exerted an anti-tumor effect in the melanoma tumor model.Part Ⅱ The role of Eomes in IL-33 regulation ’of the effector function of mouse CD4+Th9 and CD8+Tc9 cells cultured in vitro[Objective]To explore the role of Eomes in the regulation of IL-33 regulation of mouse CD4+Th9,CD8+Tc9 cells in vitro and provide theoretical basis and new strategies for the application of Th9 and Tc9 in adoptive immunotherapy.[Methods]In this study,CD4+and CD8-T cells derived from the spleen of WT and EKO mouse were purified by Microbeads.In the with or without of cytokine IL-33,cells required the polarization of CD3 mAb(5μg/ml),CD28 mAb(5μg/ml),IL-2(20U/ml),mlL-4(10ng/ml),mTGF-β(1ng/ml)and anti-IFN-γ(10μg/ml)(Th9 or Tc9).After conditioned culture for 72 hours,cells of each group were collected and cultured for 4 hours in the presence or absence of cytokine IL-33.Real-time PCR was used to analyze the expression of IFN-y and IL-9 mRNAs.At the same time,cell culture supernatant was collected to detect IFN-y and IL-9 secretion by ELISA.Cells were collected for 48 hours,and the expression of IL-9 was detected by flow cytometry.[Results]1、In the differentiation and activation of murine CD4+Tc9 cells regulated by IL-33 in vitro,deficient of Eomes gene further enhanced up-regulation of IL-9,IFN-γ,Granzyme A,TGF-β,PU.1,IRF4,CCL20,CCR6 and other molecules,and the difference was statistically significant.(P<0.05).2、In the differentiation and activation of murine CD8+Tc9 cells regulated by IL-33 in vitro,deficient of Eomes gene further enhanced up-regulation of IL-9,IFN-γ,Granzyme A,TGF-β,PU.1,IRF4,CCL20,CCR6 and other molecules,and the difference was statistically significant.(P<0.05).[Conclusion]In the process of mouse CD4+Th9 cells polarizing,deficient of Eomes gene further enhanced the up-regulation of IL-9,TGF-P,PU.1 and IRF4 expression regulated by IL-33,promoted the polarization of CD4+Th9 cells and the production of IL-9.In addition,deficient of Eomes gene can further enhance the up-regulation of the expression of IFN-y and Granzyme A mediated by IL-33,and promote the effector function of CD4+Th9 cells.Similar results were obtained during the polarization of mouse CD8+T cells to CD8+Tc9 cells,suggesting that Eomes gene deficient can promote the polarization of CD8+Tc9 cells and enhance cell-effect function.These results provide a potential idea for the application of CD4+Th9 and CD8+Tc9 cells in adoptive cell transfer immunotherapy. |
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