The Preliminary Study On IL-36β Promoting Tc9 Cell Polarization And Adoptive Cellular Immunotherapy For Tumors

Recently,adoptive cell immunotherapy(ACT)has made a significant progress.Currently,a common method in tumor adoptive T cell immunotherapy is to obtain CD8+cytotoxic lymphocyte(CD8 CTL,also known as Tcl cells),which is characterized by secreting effective cytokines such as IFN-y.Due to the strong killing effect,Tcl cells usually cause the cytokine release syndromeand(CRS).At same time,the terminal differentiation of Tel cells lead to the limited persistence of their function in vivo.Hence,adoptive cellular imunotherapy based on Tcl cells is limited in clinical therapy application.Therefore,it’s significant for improving the effect of tumor immunotherapy to design adoptive CD8+T cells with long-term survival or proliferative ability and sustained function of killing the tumor after reinfusion.In recent studies,Tc9 cells are characterized by their secretions of IL-9.IL-10,IL-21 and IL-22.Compared to Te1 cells,Tc9 cells have been reported to have a stronger inhibitory effect on tumors for its long-lasting anti-tumor effect and less prone to functional exhaustion in vivo.Therefore,Tc9 cells can be used as an ideal type of CD8+T cell differentiation for tumor adoptive immunotherapy.Finding a method that can effectively promote the differentiation of Tc9 cells has innovative significance and potential application value for tumor immunotherapy.This study was to investigate the effect of IL-363 on the differentiation of Tc9 cells,as well as to analyze the effect of IL-36β-promoting Tc9 cells in tumor adoptive immunotherapy,thereby laying a theoretical foundation for finding effective methods to promote Tc9 differentiation.Part Ⅰ Study on the effect of IL-36β on Tc9 cells differentiationObjective:To investigate the effect of IL-36P on the differentiation of Tc9 cells and to explore the biological characteristics of Tc9 cells.Method:First,we use immunomagnetic beads to purify CD8+T cells from C57BL/6j mouse.Then,under the differentiation condition of Tc9,TcO,Te1,Tc2,Tc17 or under the condition of combined cytokine such as TGF-P,IL-4 with or without IL-36p,these CD8+T cells were stimulated resoectively with anti-CD3 and anti-CD28 antibody plates.Later.the expression levels of IL-9 gene and protein in each group were analyzed by real-time quantitative PCR(QRT-PCR)and flow cytometry.Moreover,we use immunomagnetic beads to purify CD8+T cells from OT-I mouse.Then,under the condition of combined cytokine such as TGF-p,IL-4 with or without IL-36P.these CD8 T cells were stimulated resoectively with antigen-presenting cells(APC)loaded with OVA antigen peptide(SIINFEKL).Later,IL-9 protein expression levels were analyzed by flow cytometry.The levels of IL-9 and IL-10 in the cell culture supernatant were determined by ELISA.In addition,transcriptome sequencing was used to analyze the expression of related cytokines and transcription factors in Tc9 cells stimulated with or without IL-36p.Results:The results showed that IL-36P up-regulated the expression of IL-9 gene in TcO.Tc2,Tc9 and Tc17 cell subsets in different degrees.Under the condition of Tc9 cell differentiation,IL-36P significantly promoted the expression of IL-9 and IL-10 genes and protein.Transcriptome sequencing showed that IL-36[3 up-regulated the expression of IL-21,IL-22,BATF and other characteristic cytokines or transcription factors related to Tc9 cells.It also promoted the expression of IFN-y,FasL,perforin,GzmA,GzmB,GzmC and other effector molecules,but down-regulated the expression of Foxp3.Conclusion:IL-36P can significantly promote the expression of Tc9 cell-associated cytokines and transcription factors,as well as up-regulate the expression of some effector molecules.Iii short,IL-36[3 promotes the differentiation and function of Tc9 cells.Part Ⅱ Preliminary study on the role of IL-36β-induced Tc9 cells in adoptive immunotherapy of tumorsObjective:To investigate the effect of IL-36β-induced Tc9 cells on tumor adoptive immunotherapy and to analyze its possible mechanism.Methods:B16-OVA cells were subcutaneously inoculated on the outside of the abdomen of C57BL/6j mice with CD45.1 or CD45.2 background to construct a tumor-bearing mouse model.Then,cyclophosphamide was intraperitoneally injected to remove lymphocytes from mice on the 5th day.and bone marrow-derived dendritic cells(BMDC)loaded with OVA antigen peptide(SIINFEKL)were intraperitoneally injected on the 6th day.At the same time,OVA antigen-specific CD45.2+Tc9 cells and other CD8+T cell subsets stimulated in vitro were used for adoptive treatment.Observed the growth of tumor and the survival of tumor-bearing mice every 2 days,and draw the curves of tumor growth and mouse survival.After 20 days of cell therapy,the ratio of exogenous CD8+T cells(CD45 CD8 CD45.2+)and endogenous CD8+T cells(CD45 CD8 CD45.2")were detected by flow cytometry in tumor tissues,spleens and tumor draining lymph nodes of tumor-bearing mice.The expression levels of IL-9 and IFN-y in exogenous and endogenous CD8+T cell subsets and immune checkpoint molecular PD-1,CD244 were also detected.Results:Compared with Tc9 cells induced by TGF-B combined with IL-4,IL-363-induced Tc9 cells can inhibit tumor growth more effectively and prolong the survival of tumor-bearing mice.A higher proportion of exogenous CD8+T cells were detected in the tumor tissue,spleen and tumor draining lymph nodes of the adoptive treatment group of IL-36[3-induced Tc9 cells.There was no significant difference in IL-9 and IFN-y expression of exogenous and endogenous CD8+T cells between the classical Tc9 cells and IL-36β-induced Tc9 cells adoptive treatment groups.There was no significant difference in PD-1 and CD244 expression of endogenous CD8 T cells between the two groups of tumor-bearing mice.However,compared with the classical Tc9 adoptive treatment group,PD-1 and CD244 expression of exogenous CD8+T cells had a relatively low level of expression in the IL-36β-induced Tc9 adoptive treatment group.Conclusion:Compared to Tc9 cells induced by TGF-β combined with IL-4,,IL-36β-induced Tc9 cells possess more significant tumor adoptive therapeutic effects.After adoptive transfer,it has a higher level of distribution in tumor-bearing mice,and expresses lower levels of immune checkpoint molecules such as PD-1 and CD244.