IL-36? Promotes CD8~+ T Cell Function By Activating MTORC1 And Enhances CD8~+ T Cell-mediated Antitumor Immune Responses

Part ?:The mechanism of IL-36? activating CD8+ T cells dependent on mTORC1 pathwayObjective:CD8+T cells are the most important anti-tumor efifector cell population in adaptive immune response.Interleukin-36(IL-36)includes three agonists,IL-36?,IL-36?and IL-36?,which act on the same receptor IL-36R.It has been found that IL-36? has a significant promoting effect on CD8+T cell.This section aims to further clarify whether IL-36? can promote CD8+T cell activation,and on the basis of this,IL-36? is used to explore the molecular mechanism of CD8+T cell activation mediated by IL-36/IL-36R signaling.Methods:Primary CD8+T cells,isolated from C57BL/6j mice and labeled or unlabeled with CFSE(5-(and-6)-Carboxyfiuorescein Diacetate Succinimidyl Ester),were stimulated by plated anti-CD3 and anti-CD28 elicited monoclonal antibodies,either alone or in combination of IL-36?,IL-2 and IL-12.The expression of early activation markers CD69 and CD25 were determined by flow cytometry,and we analyzed CD8+T cell proliferation by measuring CFSE.ELISA method was used to determine IL-2 and IFN-? levels in culture supernatants.Flow cytometry or western blot was used to determine the phosphorylation of ribosomal protein S6(p-S6),a downstream marker of mTORC1(mammalian target of rapamycin,mTOR)activation,phosphorylation of Akt(p-Akt)and degradation of I?B.With or without mTORC1 specific inhibitor Rapamycin,inhibitor against phosphoinositide 3-Kinase(PI3K)or Inhibitor of Nuclear Factor Kappa-B Kinase,p-S6 in CD8+T cells was respectively determined by flow cytometry or western blot.The p-S6 levels,cell activation,the expression of IL-2 and IFN-?,and cell proliferation in CD8+T cells,which were derived from Myeloid Differentiation Factor 88(MyD88)-deficient mice,after stimulation,were analyzed.Results:The results showed that IL-36? could significantly promote the activation and proliferation of CD8+T cells.At the very early stage of stimulation,IL-36? can induce the production of IL-2 and IFN-?.IL-36p also significantly promoted the phosphorylation of the downstream signal molecule S6 of mTORC1 in CD8+T cells,while rapamycin completely inhibited IL-36p-mediated mTORC1 activation in CD8+T cells,and simultaneously inhibited cell growth,proliferation and secretion of IL-2 and IFN-?.IL-36? upregulated p-Akt,but p-S6 was significantly inhibited in the presence of PI3K inhibitors.The degradation of I?B could be significantly induced by IL-36p,while inhibition of IKK activity led to down-regulation of S6 phosphorylation mediated by IL-36p.MyD88 deficiency caused IL-36p-mediated S6 phosphorylation of CD8+T cells to be almost inhibited,and mTORC1 was inactivated due to MyD88 deficiency,which inhibited IL-36?-mediated cell growth,proliferation,and cytokine production.Conclusion:This study demonstrated that IL-36p activated the mTORC1 signaling pathway and mediated activation and proliferation of CD8+T cells through the PI3K/Akt,IKK,and MyD88 pathways.Part ?:The effect of IL-36p promoting CD8+TILs anti-tumor immune responsesObjective:CD8+ tumor-infiltrating lymphocytes(TILs)are often functionally suppressed in a state of exhaustion.Recovering or boosting antitumor function of CD8+ TILs by appropriate means is considered as an effective pathway to elicit tumor immune response and eliminate tumors.This section aims to investigate the effect of IL-36p on the function of CD8+ TILs in the tumor microenvironment and its inhibitory effect on tumor growth.Methods:First,4T1 breast cancer cells and B16 melanoma cells were adopted to respectively generate 4T1-vector(4T1-vec),B 16-vector(B16-vec)as the controls and 4T1-IL-36?,B16-IL-36? over-expressed mouse IL-36?.These cell lines were injected into BALB/c mice or C57/BL6 mice,then tumor growth was monitored and mouse survival was observed.On day 23 of B16-vec and B16-IL-36p tumor growth,the frequency of CD8+ TILs,nuclear proliferating antigen Ki67 expression and IFN-? secretion were analyzed by flow cytometry.Meanwhile,the frequencies of CD45+ population,NK and??T cells in the tumor microenvironment,as well as the frequency of Myeloid-Derived Suppressor Cells(MDSCs)and the expression of their major histocompatibility complex class ? molecules(MHC-?)were determined.The level of IFN-? released by tumor antigen-specific CD8+ T cells in 4T1-vec and 4T1-IL-36? tumor-bearing mice was measured by ELISA method.Results:The results showed that IL-36p obviously inhibited the growth of breast cancer 4T1 and melanoma B16.Meanwhile,IL-36p significantly prolonged the survival of melanoma B10 tumor-bearing mice.IL-36p signiflcantly increased the number of CD8+TILs,promoted the proliferation of CD8+ TILs,up-regulated IFN-? expression in CD8+TILs,and enhanced the adaptive tumor antigen-specific CD8+ T cell immune response.At the same time,the frequencies of CD4+ T,NK and ??T cells in CD45+ cells were increased by cytokine IL-36?.Additionally,IL-3 6pdown-regulated Neutrophil Myeloid Derived-Suppressor Cells(NMDSCs),while upregulated the expression of MHC-?molecules in NMDSCs and mononuclear myeloid-derived suppressor cells(MMDSCs)expression on MMDSCs.Conclusion:Cytokine IL-36? could inhibit tumor growth and prolong the survival of tumor-bearing mice.In addition,it couldtransform the tumor immune microenvironment,promote the function of CD8+ TILs,and enhance antitumor antigen-specific CD8+ TILs responses.