Study On The Mechanism Of IL-36? Promoting Tc9 Cell Polarization And Adoptive Immunotherapy Of Tumor

Under the induction of different cytokines,CD8+T cells can differentiate into subgroups with different characteristics and functions,including Tc1,Tc2,Tc9,and Tc17.Among them,Tc1,which is type I CD8+cytotoxic T cells(CD8+CTL),has high cytotoxicity,while Tc9 and Tc17 cells exhibit low cytotoxicity in vitro.At present,adoptive cell therapy(ACT)with type ICD8+CTL,has shown good clinical therapeutic value.Recent studies have revealed that adoptive immunotherapy with tumor-specific Tc9 cells can produce a more durable and effective anti-tumor response in mouse melanoma than traditional Tc1 cells,which is related to the long-term survival characteristics of Tc9 cells and its ability to transforming to Tcl-like cell secreting IFN-? and Granzyme B.Therefore,inducing Tc9 cells with excellent properties and applying them to tumor adoptive immunotherapy is a valuable approach to cancer treatmentThis study is aimed to explain the effect of IL-36? on Tc9 polarization,investigate its mechanism of promoting Tc9 polarization,and explore the role and mechanism of IL-36?-induced Tc9 cells in tumor adoptive immunotherapyPart ? Study on IL-36y promoting Tc9 cells polarization and its mechanismObjective:To study the promoting effect of IL-36? on Tc9 cell polarization and further explore its potential molecular mechanismMethods:Mouse CD8+T cells were sorted by microbeads and cultured in vitro for 12 hours under three differentiation conditions of Tc1,Tc9(TGF-? and IL-4)and Tc9+IL-36?(TGF-?,IL-4 and IL-36?).Then,Real-time quantitative PCR(qRT-PCR)was used to detect the levels of Tc9-related cytokines in these cells.Besides,CD8+T cells from C57BL/6j mice were stimulated with anti-CD3 and anti-CD28,then cultured with different combinations of TGF-?,IL-4 and IL-36?,or cultured with different concentrations of IL-36? for 48 hours in vitro,and the expression of IL-9 and IL-10 was finally analyzed by flow cytometry and ELISA.Similarly,CD8+T cells from OT-I mice were stimulated with OVA257-264 peptide by antigen-presenting cells in vitro,and analyzed by flow cytometry and ELISA to detect the expression of IL-9 and IL-10.In addition,different concentrations of STAT5,STAT6,and NF-?B inhibitors were added during in vitro culture,and the expression level of IL-9 was detected by ELISA in IL-36?-inuced Tc9 cells.Finally,transcription factors BATF and BATF3 were analyzed by qRT-PCRResults:The cytokine IL-36? can significantly increase the expression of cytokines IL-9,IL-10 and IL-17a.The addition of IL-36? does not increase the expression of IL-4,indicating that IL-36? directly promotes Tc9 cells polarization Stimulated by anti-CD3 and anti-CD28 or antigen-presenting cells in vitro,IL-36? can replace IL-4 in classical differentiation condition,and significantly increase the expression and secretion of IL-9.Besides,the expressions of IL-9 and IL-10 were significantly increased,when CD8+T cells cultured under classical differentiation condition added with IL-36?.When STAT6,STAT5 and NF-?B signaling pathways were blocked respectively,the IL-36? promoting effect on Tc9 polarization was inhibited.In addition,IL-36? promoted the expressions of transcription factors BATF and BATF3Conclusion:The cytokine IL-36? can significantly promote Tc9 cells polarization and increase the expression of related cytokines.This effect is dependent on STAT6,STAT5,NF-?B pathway and related to transcription factor BATF and BATF3Part ? Study on the role and mechanism of IL-36y-induced Tc9 cells in tumor adoptive immunotherapyObjective:To study the effect and mechanism of IL-36?-induced Tc9 cells on adoptive immunotherapy in mouse melanomaMethods:CD45.1+ mice were subcutaneously inoculated with B 16-gOVA cells to establish a mouse adoptive immunotherapy model.On the fourth day of tumor injection,cyclophosphamide(CTX)was injected intraperitoneally for temporary clearance of lymphocytes in mice.On the 5th day,Tc1,Tc9 and IL-36?-induced Tc9 cells,which have cultured for 48 hours in vitro,were intraperitoneally injected into mice.Then,the size of the tumors was measured and the survival of the mice was observed every other day from the 3rd day of tumor injection.After 14 days of adoptive immunotherapy,the exogenous and endogenous CD8+T cells in the spleen,tumor draining lymph nodes(TDLN)and tumor tissue of tumor-bearing mice,and its expressions of memory phenotype,immune checkpoints were detected by flow cytometryResults:Compared with traditional Tc9 cells,IL-36?-induced Tc9 cells can significantly inhibit the progression of melanoma.After 14 days of adoptive immunotherapy,the proportions of IL-36?-induced Tc9 cells in mouse were increased significantly,compared with traditional Tc9 cells.IL-36?-induced Tc9 cells expressed lower levels of immune checkpoints CD244,PD-1,and KLRG-1 in vivo.In addition,the memory phenotype of IL-36?-induced Tc9 cells in vivo was basically consistent with that of traditional Tc9 cells,and they both expressed high levels of IFN-? and Granzyme B after restimulated with antigenConclusion:Compared with traditional Tc9 cells,IL-36?-induced Tc9 cells have better tumor adoptive immunotherapy effects,and maintain a higher number in vivo and low expression of immune checkpoint CD244,PD-1 and KLRG-1.IL-36?-induced Tc9 cells maintain memory phenotype of traditional Tc9 cells,and Tcl-like characteristics of high levels of IFN-? and Granzyme B.Studying the role and mechanism of IL-36y-induced Tc9 cells in tumor adoptive immunotherapy can lay a solid theoretical foundation for inducing Tc9 cells with excellent performance and applied to tumor adoptive immunotherapy.Compared with traditional Tc1 cell,IL-36?-induced Tc9 cells in adoptive tumor immunotherapy may have greater clinical treatment prospects,which deserves further exploration.