The Preliminary Study On IL-33 Mediating Anti-tumor Immune Response By Regulating The Effector Functions Of CD4~+Th9 Cells And The Expression Of IL-9

Tumor microenvironment is a complex environment to the survival of tumor cells,which is a comprehensive state system composed of tumor cells,stromal cells,infiltrating immune cells and a series of molecules.The effect of the body's tumor immune responses is determined by the ingredients in tumor microenvironment,the interactions between tumor cells,and the number,types and functions of txumor-infiltrating immune cells,while the cellular immune response mediated by CD4+helper T cells(Th cells)and CD8+ cytotoxic T l-yrphocytes(CTLs)is the basis of anti-tumor immunity.A variety of immunosuppressive factors produced by tumor microenvironment can lead to CD4+Thl cells and CD8+ CTLs in a state of impotency or exhaustion,and cause the body with no effective antitumor immune response.Therefore,it has become a research focus of immunotherapy to looking for a new molecular and new method,which can activate the tumor infiltrating lymphocytes(TILs)and change the state of immunosuppressive of tumor microenvironment.IL-33,the 11th member of Interleukin-1 family,mainly involved in Th2 immune response by binding to the receptor complex consisted of its receptor ST2 and IL-1 receptor accessory protein.It has also demonstrated that IL-33 mediates differentiation of Thl cells and production of IFN-y.However its mechanism of anti-tumor immune response is still unknown.The IL-9 gene of human,which is mapped on chromosome 5,in 5q31-q35 region,and the IL-9 gene of mouse shares a 55%amino acid homology.IL-9 binds to its receptor IL-9R and plays a role by inducing phosphorylation of JAK1 and JAK3,activating STAT1,STAT3,STAT5 forming homodimers,promoting expression of a series of genes in nucleus.CD4+Th9 cells subset could secrete cytokine of IL-9 to mediate variety immune response.Many studies found that antigen-specific Th9 cells induced CCL20/CCR6 signaling pathway to generate stronger antitumor effect in adoptive immunotherapy comparing to Thl and Thl7 cells.However it is unclear that the direct effect and mechanism of CD4+Th9 cells and IL-9 in tumor immune response.Our previous studies have found,IL-33 synergized with IL-12 signaling can induce CD8'T cells producing IFN-y,enhance the anti-tumor effect function of CTLs,and reduce the tumor growth;IL-36?stimulated mouse CD 8+T to secrete abundant IL-9 in vitro culture.Based on these findings,we investigated the influence of IL-33 on activation,differentiation and function of CD4+T cells purified from human PBMCs or mouse spleen cells under Th9-polarization conditions.As a regulatory factor for anti-tumor immune response of CD4+Th9 cells,such studies will provide theoretical basis and novel strategy for applying IL-33 in tumor adoptive cellular immunotherapy.And then we further studied the expression of IL-9 during tumor progression and the effect of IL-9 on tumor growth in different tumor models with IL-9 knockout mouse and discussed whether IL-33 could mediate anti-tumor immune response by inhibiting the expression of IL-9 in tumor microenvironment and its possible regulatory mechanism.The above researches may provide new methods and train of thought in tumor immunotherapy.Part ? IL-33 can regulate the effector functions of human andmouse CD4+Th9 cells in vitroObjective:To investigate whether recombinant human/mouse IL-33 can directly effect on human/mouse CD4+T cells to enhance the activation,differentiation and function of human/mouse CD4+Th9 cells and promote secretion of its cytokines,and provide theoretical basis for clinical tanslational research in adoptive cellular immunotherapy of CD4+Th9 cellsMethod:1)Collect human CD4+T cells by positive selection of CD4 magnetic beads from healthy volunteers' PBMCs,and then CD4+T cells were stimulated with different combination of cytokines in vitro:CD3 mAb(100 ng/ml)+CD28 mAb(200 ng/ml)+IL-4(10ng/ml)+TGF-?(1ng/ml)(Th9 condition),CD3 mAb(100 ng/ml)+CD28 mAb(200 ng/ml)+IL-4(10 ng/ml)+TGF-?(1 ng/ml)+IL-33(30 ng/ml).96h later,cultured cells were re-stimulated for another 4h or 24h with/without IL-33(30 ng/ml)in the present of CD3 mAb(100 ng/ml).2)Purify mouse CD4+T cells by positive selection of CD4 magnetic beads from mouse spleen cells,and then CD4+T cells were stimulated with different combination of cytokines in vitro:CD3 mAb(5 ?g/ml)+CD28 mAb(2.5(?g/ml)+IL-4(10 ng/ml)+TGF-?(1 ng/ml)(Th9 condition),CD3 mAb(5 ?g/ml)+CD28 mAb(2.5 ?g/ml)+IL-4(10 ng/ml)+TGF-P(1 ng/ml)+IL-33(50 ng/ml).Cultured cells were re-stimulated for another 4h or 24h with/without IL-33(50 ng/ml)in the present of CD3 mAb(5?g/ml)96h later.Total RNA was extracted use the RNeasy mini kit and assayed by Real-time PCR to detect the expression of IL-9,interferon-y(IFN-y),granzyme A,IL-17A,PU.1,IRF4,CCL20,CCR6 and other molecules in mRNA levels;The expression of IL-9 and IFN-? was detected by Flow cytometry.Result:1.IL-33 significantly increased the expression of IL-9,IFN-?,Granzyme A,IL-17A,transcription factor PU.1 and IRF4,chemokine CCL20 and chemokine receptor CCR6 of human CD4+ Th9 cells.2.IL-33 can significantly enhance the expression of IL-9,IFN-? and Granzyme A of purified mouse CD4+ Th9 cells.Conclusion:Cytokine IL-33 can enhance the effector functions of CD4+Th9 cells,promote anti-tumor immune response.This may provide a new method and train of thought for tumor adoptive cellular immunotherapy,and have potential clinical application value.Part ? IL-33 can mediate anti-tumor immune response by regulating the expression of IL-9 in tumor microenvironmentObjective:To investigate whether L-33 can mediate anti-tumor immune response by regulating the expression of IL-9 in tumor microenvironmentMethod:C57BL/6 wild type(WT)mice and IL-9 knockout mice(5 mice for each group)were challenged with 2×105 B16 cells or B16-IL-33 cells i.d.And Mice were monitored for tumor growth every other day.Tumor masses of each group were removed at early,middle and later period during tumor growth,total RNA was extracted and assayed by Real-time PCR to detect the expression of IL-9 in mRNA levels.Result:1.During tumor progression,tumor of C57BL/6 WT mice grown faster comparing to IL-9KO mice In B16 tumor-bearing mouse;however,there is no difference between C57BL/6 WT mice and IL-9KO mice in B16-IL-33 tumor-bearing mouse.2.IL-9 sustained high expressed in B16 tumor microenvironment,and low expressed in B16-IL-33 tumor microenvironment.Conclusion:In vivo,the expression level of IL-9 in tumor microenvironment is correlation with tumor development.However,IL-33 might inhibit the expression of IL-9 in vivo to act anti-tumor immune response.The exactly regulatory mechanism remains to be further studied.