Complex Interplay Between MTORC1 And MTORC2 Regulates NK Cell Maturation And Effector Function

Natural killer cells?NK?are an important component of the body's natural immune system and play a key role in monitoring the early malignant transformation of tumors,eliminating viruses and infecting cells,and regulating immune system responses.Disability of NK cells leads to tumor deterioration and increased metastasis,while excessive activation of NK is a major cause of some autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.Therefore,exploring the mechanisms involved in the regulation of NK cell development and function is of great significance for understanding the occurrence of various diseases,providing targeted research directions and treatment strategies.The regulation of NK cell development and function is mainly dependent on the regulation of exogenous and endogenous factors:the exogenous factors mainly include IL-2,IL-15,IL-12,IL-18,TGF-?and other cytokines as well as amino acid,glucose and other metabolites;the endogenous factors mainly include some transcription factors,such as foxo1,GATA-3,TOX,Tbx-21,Eomes,and IRF2 et.al.During recent years,researchers have found that the development and function of NK cells are closely related to their metabolic state.During the development of NK cells,their metabolic level will gradually decrease,and when NK cells are stimulated by cytokines,tumors or viruses,their metabolic level will be significantly enhanced.On the contrary,inhibition of NK cells metabolism will lead to impaired development and activation.In mammalian cells,the most important pathway regulating cell metabolism is the mTOR signaling pathway,which consists of mTORC1?a protein complex with Raptor as the core?and mTORC2?a protein complex with Rictor as the core?.The mTOR signaling pathway can respond quickly to the changes of extra-cellular environment and regulate the process of intracellular nutrient metabolism and substance synthesis effectively to ensure the survival and growth of cells.It has been found that abnormalities of the mTOR signaling pathway are involved in the development of a variety of human diseases,including tumors,obesity,type 2 diabetes,and neurodegenerative diseases.In NK cells,previous studies have found that treatment with rapamycin,a small molecule inhibitor of mTORC1,or knockout of the mTOR signaling pathway entirely will reduce the responsiveness of NK cells to cytokine IL-15 and impair the development and function of NK cells.However,treatment with rapamycin inhibitors can affect the activity of mTORC1 in various cells,which may have an indirect effect on NK cells,and the specificity of rapamycin to mTORC1 pathway has also been questioned.Therefor,rapamycin treatment can not accurately reflect the effects of mTORC1 on NK cell development and function under physiological conditions.Besides,the overall knockout of mTOR inhibits the activity of mTORC1 and mTORC2 at the same time.Therefore,there are still many key questions that remain unclear,for example,what is the role of mTORC2signaling pathway in NK cells,and what is the mechanism involved;what are the similarities and differences between mTORC2 and mTORC1 in the regulation of NK cells,and whether there is inter-connection between them.Based on the questions above,in the present study,we constructed transgenic mouse models that specifically knocked out mTORC1 or mTORC2 in NK cells,and studied their roles and crosstalks in the regulation of NK cell development and function as well as the mechanisms involved.Our study provides a new strategy for anti-tumor immunotherapy targeting the mTOR signaling pathway in NK cells.Methods and results:In the present study,we crossed the Vav1-Cre and Ncr1-iCre tool mice with Rictor^fl/fll/fl or Raptor^fl/fl mice to construct mice models capable of specifically knocking out Rictor protein in NK cells(Vav-Cre⁺/Rictor^fl/fl and Ncr1-iCre⁺/Rictor^fl/fll/fl mice,termed as Rictor^?/?and Rictor^?NK,respectively),or Raptor protein in NK cells(Ncr1-iCre⁺/Raptor^fl/fl mice,termed as Raptor^?NK),or half-knocking down Raptor protein based on the completely loss of the Rictor protein in NK cells(Ncr1-iCre⁺/Rictor^fl/fl/Raptor^fl/wt mice,termed as Rictor^?NKNK Raptor^fl/+).Based on the transgenic mice models,we carried out a series of in vitro and in vivo experiments with flow cytometry,immunoblotting or qRT-PCR reaction,and obtained the following results:1.mTORC2 signaling pathway is involved in maintaining NK cell development and maturation.We found that the lineage-specific differentiation of NK cells in bone marrow from Rictor^?/?mice is hindered,and the proportion and number of NK cells in the spleen of Rictor^?/?or Rictor^?NK mice were significantly reduced.In each organ,the development of NK cells was significantly blocked,especially the proportion of the most mature CD27^-CD11b⁺cell subsets was significantly reduced.2.mTORC2 signaling pathway negatively regulates the effector functions of NK cells.The spleen cells or sorted NK cell subsets from Rictor^?/?or Rictor^?NK mice were co-cultured with target tumour cells respectively in vitro.Flow cytometry showed that the CD107a expression level on Rictor-deficient NK cells was higher and fluorescence tests showed that Rictor-deficient NK cells had higher killing efficiency against target cells in vitro.Consistently,we also performed tumour clearance assay in vivo,and Rictor^?/?or Rictor^?NK mice showed higher clearance efficiency compared with the control.In addition,Rictor-deficient NK cells also expressed more cytotoxic molecules and metabolism related indicators.3.mTORC1 signaling pathway positively regulates both the development and functions of NK cells.The proportion and number of NK cells in Raptor^?NK mice increased significantly in the bone marrow,but decreased in the spleen.However,in each organ,the developmental process of NK cells was blocked,showing a significant decrease in the proportion of the mature CD27^-CD11b⁺subpopulations cells,while an increase in the proportion of immature CD27⁺CD11b^-subpopulations.In terms of functions,Raptor deficiency did not affect the proportion of NK cells that secreted IFN-?,but reduced the secretion level in individual cells.In addition,the expression level of CD107a was significantly decreased when NK cells were stimulated by target cells in vitro.Consistently,the ability of Raptor^?NKNK mice to clear tumor cells in vivo was impaired,and the expression of toxic molecules and metabolic related indicators in NK cells also decreased significantly.4.The crosstalks between mTORC1 and mTORC2 signaling pathways in NK cells.We found when stimulation of normal human and murine NK cells with the cytokine IL-15,the activity of mTORC1 and mTORC2 signaling pathways in the cells both increased at first and then decreased following continuous stimulation.In addition,Raptor-deficient NK cells showed decreased expression of p-AKT^ser473,which was due to the decreased expression of CD122 on the surface,resulting in decreased responsiveness of NK cells to IL-15 stimulation.In contrast,in Rictor-deficient NK cells,we found the expression level of p-S6 was significantly increased,and the increase would be more pronounced when stimulated with IL-15.5.mTORC1 and mTORC2 signaling pathways have non-redundant regulation on NK cell development.To further explore the corsstalks between mTORC1 and mTORC2 signaling pathways,we constructed the Rictor^?NKRaptor^fl/+mice model.We found relative to Rictor^?NK mice,the proportion and number of NK cells in Rictor^?NKRaptor^fl/+mice were further reduced and the development was further blocked,which suggested that mTORC1 and mTORC2 had relatively independent regulatory mechanism in NK development.To elucidate this mechanism,we examined the expression levels of the transcription factors Tbx21 and Eomes,which play a major role in the regulation of NK cell development,in NK cells from Rictor^?/?,Raptor^?NK,and Rictor^?NKRaptor^fl/+mice.We found in Rictor-deficient NK cells,the expression level of Tbx21 was significantly decreased,while Eomes was unchanged;in Raptor-deficient NK cells,the expression levels of Tbx21 and Eomes were both decreased;in Rictor^?NKRaptor^fl/+NK cells,the expression of Tbx21 and Eomes were consistent with that in Rictor-deficient NK cells.6.mTORC2 inhibits the effector function of NK cells through suppressing the activity of mTORC1 signaling pathway.To investigate whether the inhibition of mTORC2 on NK cell functions and metabolism was resulted from its suppression on mTORC1 activity,we examined the functions and metabolism of NK cells in Rictor^?NKRaptor^fl/+mice.The results showed that the functions and metabolism of NK cells in Rictor^?NKRaptor^fl/+mice were significantly lower than those in Rictor^?NK mice,which were basically consistent with control.However,the expression of CD107a still showed an increasing trend compared to the control.Besides,we also observed a similar phenomenon in Rictor-deficient NK cells after treatment with low concentration of rapamycin,which exhibited comparable level of mTORC1 activity to that in WT NK cells.7.The mechanism of mTORC2 inhibiting mTORC1 activity in NK cells.First of all,we found the PI3K-AKT signaling pathway was not involved in Rictor's negative regulation of mTORC1 activity.Although the expression of CD122 on the surface of Rictor-deficient NK cells did not change,the activity of p-STAT5 signaling path way was significantly increased.In addition,treatment of Rictor-deficient NK cells with the specific p-STAT5 inhibitor could reduce the expression of p-S6 significantly as well as the effector functions.To further elucidate how p-STAT5 promote mTORC1 activity in NK cells,we detected the expression of SLC7A5 on cell surface,which is transcriptionally regulated by p-STAT5 while regulates mTORC1 activity in cells.The results showed that SLC7A5 was increased significantly on Rictor-deficient NK cells and treatment with the p-STAT5inhibitor could reverse the increase.Consistently,we inhibited SLC7A5 with BCH in Rictor-deficient NK cells and found mTORC1 activity was also reversed.Conclusions:1.mTORC1 and mTORC2 signaling path way regulate NK cell development in a relatively cooperative and independent way,through regulating the expression of Tbx21and Eomes.2.mTORC1 promotes the effector functions and metabolism of NK cells,while mTORC2 is on the contrary.3.In NK cells,mTORC1 regulates mTORC2 activity through maintaining the CD122-IL-15 path way,while mTORC2 inhibites mTORC1 activity by limiting p-STAT5induced SLC7A5 expression,resulting in the suppression on effector functions and metabolism.In conclusion,our study revealed the crosstalks between mTORC1 and mTORC2signaling pathways in NK cells,elucidating the mechanisms by which mTORC1 and mTORC2 are dynamically regulated during NK cell development and activation,and broadening the knowledge about interactions between mTOR and STAT5 in NK cells.In particular,our study proposes a new strategy for the anti-tumor immunotherapy of NK cells by targeting at the inhibition of mTORC2 to enhance the effector functions in NK cells.