| Natural killer cells(NK cells)are one of the most important innate lymphoid cells(ILC)in vivo,which play an essential role in the immnosurveillance and clearance of tumors.It has been confirmed that NK cells can kill several hematopoietic system deri ved tumors in vitro and clear some transplantation or spontaneous tumors effectively in mice.Besides,an 11-year following up study has found that people with relatively low natural cytotoxicity in peripheral blood are faced with higher risk of tumor.As a result,NK cells have been generally focused as a promising target in anti-tumor immunotherapy.In anti-tumor immune response,NK cells can recognize and be activated by the ?missing self” mechanism.On one hand,when contact with tumor cells directly,NK cells can secret cytotoxic molecules,such as perforin and Granzyme B,to lyse and kill tumors.On the other hand,NK cells can secret cytokines,mianly IFN-?,to be engaged in the regulation of anti-tumor responses.Functions of NK cells are controlled b y various intracellular and extracellular factors,including transcriptional factors and cytokines.However,recent studies have confirmed that functions of NK cells are tightly connected with their metabolic status.When NK cells are activated under stimu lation,their metabolic level are increased significantly,and the metabolic state is transited from oxidative phosphorylation into glycolysis.Besides,it has been reported that the metabolism of NK cells is under the direct control of mTOR,especially mTORC1,signaling pathway.Though NK cells are effective at killing tumor cells,the ability of NK cells clearing solid tumors in vivo is always questioned.It is mainly because that at the tumor genesis area,the tumor microenvironment has strong immunosuppression effect,which is one of the reasons that why tumor cells can escape from immnosurveillance or be tolerant to immune responses.In current study,we learned from the work of Ende Zhao(Nat Immunol,2016)and built a model of tumor microenvironment in vitro.We explored the influence of microenvironment on NK cells functions and focused on the mechanism behind the influence.We hope that our study can raise up new strategies for targeting at NK cells in anti-tumor immunotherapy.Methods:(1)We cultured K562 constutively for 3 days and made the tumor medium by freezing and thawing the K562 cells contained medium for several times.Besides the normal medium,we set two control groups additionally,the control-1 referred to as the fresh medium containing K562 cells and the control-2 referred to as the supernatant medium after culturing K562 for 3 days.All the four medium group went through the same freezing and thawing cycles.(2)We pre-treated NKL cells by culturing them in the normal,control-1,control-2 or tumor medium for 24 h.Then we directly detected the expression of perforin,granzyme B,CD69 or NKG2 D of NKL cells or stimulated the NKL cells with PMA or K562 for another 6h to detected the expression of IFN-? by flow cytometry(FCM)or ELISA.Besides,we also co-cultured the pre-treated NKL cells with K562 cells and measured the expression of CD107 a on NKL cells or the apoptotic status of K562.(3)We measured OCR and ECAR of the pre-treated NKL cells by Seahorse XF system and the expression of P-S6 or P-AKT(ser473)in them by FCM.(4)We adjusted PH of the tumor medium back to the normal level or added glucose,glutamine or leucine into the tumor medium,and after culturing NKL cells in the adjusted tumor medium for 24 h,we detected the expression of P-S6 in them.(5)We stained the pre-treated NKL cells with TMRE to measure the mitochondrial membrane potential,Mito Tracker Green to measure the content of mitochondria,DCF-DA and MitoSox to measure the ROS and CYTO-ID to measure the autophagic level.(6)We treated the pre-treated NKL cells with ROS scavengers,including GSH,NAC and MitoTempol,for another 1h and measured the ROS level,the expression of P-S6 and the secretion of IFN-?.Results:(1)Functions of NK cells are inhibited in the simulative tumor microenviromentWe found that the control-2 medium and the tumor medium inhibited the secretion of IFN-? and reduced the expression of cytotoxic molecules,perforin and GranzymeB.When co-cultured with K562 in vitro,the control-2 medium and the tumor medium cultured NKL cells expressed lower CD107 a,and exhibited decreased cytotoxicity against K562,however their expression of the activating receptors CD69 and NKG2 D were normal.(2)The simulative tumor microenvironment can inhibit metabolism and mTORC1 signaling pathway in NK cells.We found the level of OXPHOS and glycolysis in NKL cells were both reduced when cultured in the control-2 medium and the tumor medium.As mTOR signailing pathway acts as the cellular regulator of metabolism,we found the activity of mTORC1,not mTORC2,in NKL cells was reduced significantly in the control-2 medium and the tumor medium.(3)The reduction of mTORC1 activity in NK cells was not due to the changes of nutritional or environmental factors of the simulative tumor microenvironment.We found that the reduced expression of P-S6 of NKL cells in the control-2 medium and the tumor medium was not rescued after adjustment of PH back to normal,adding glucose or amino acid.(4)The simulative tumor microenvironment impaired functions of NK cells through ROS dependent mTORC1 inhibition.In our study,we found the NKL cells that were cultured in the control-2 medium and the tumor medium exhibited reduced mitochondrial membrane potential,increased mitochondrial content,increased total or mitochondria specific ROS level and inhibited autophagic level.Besides,when we cleared the ROS in the control-2 medium and the tumor medium cultured NK cells with several ROS scavengers(NAC,GSH,mitotempol),we found that the activity of mTORC1 signaling pathway was rescued to the normal level.In consistent,after clearance of ROS,the secretion of IFN-? by NKL cells were also rescued.Conclusions:(1)The simulative tumor microenvironment can inhibit the secretion of IFN-? and cellular cytotoxicity of NK cells.(2)The simulative tumor microenvironment can inhibit metabolism and mTORC1 signaling pathway in NK cells.(3)The reduced mTORC1 activity was not resulted from the changes of PH,glucose and amino acid in the simulative tumor microenvironment.(4)The simulative tumor microenvironment can disrupt function of mitochondria and induce the accumulation of ROS by impeding autophagy.(5)The simulative tumor microenvironment inhibits NK cell functions through ROS dependent mTORC1 inhibition... |
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