The Mechanism Of IL-15 Conferring Resistance In NK Cells Against Oxidative Stress Within The Tumor Microenvironment

Objective:Cancer is the most life-threatening disease currently,apart from traditional approaches like surgery,chemotherapy,radiotherapy and targeted therapy,immunotherapy is now becoming the most potential therapeutic method against cancer.As one major branch of immunotherapy,the adaptive immune cell therapy showed less clinical efficacy in solid tumor,in spite of its impressive clinical outcomes in patients with hematological malignances.The abundance of reactive oxygen species（ROS）within the solid tumor microenvironment could dampen the infiltration and anti-tumor immune responses of immune cells.Natural killer（NK）cells are the major innate lymphocytes that can recognize and kill tumor cells in the early stage.Interleukin-15（IL-15）is important for the development and activation of NK cells.In the present study,we tried to explore the mechanism of how IL-15 infect the NK cell’s tolerance against oxidative stress.Furthermore,we also evaluated the prognostic value of NK cell infiltration and IL-15gene signature in NSCLC cohort.Thus,our study provided a new therapeutic perspective for the clinical application of NK cell-based immunotherapy in NSCLC.Methods:1. After 48 hours stimulation of IL-2 or IL-15,the cytotoxicity of NK cells against K562 was evaluated by Cr-51 release assay,and the degranulation and IFNγproduction was measured using flow cytometer.The Cr-51 release assay,degranulation assay,intracellular IFNγand ROS staining were carried out in the presence or absence of H₂O₂treatment,in aim to compare NK cell’s capacity to tolerate oxidative stress after IL-2 or IL-15 stimulation.2. In order to investigate the mechanism of how IL-15 improve NK cell’s resistance against oxidative stress,transcriptomic analysis of publicly available sequencing data was performed using gene set enrichment analysis（GSEA）.The changes of thioredoxin system in IL-15 primed NK cells were evaluated by q PCR,Western Blot,flow cytometry and immune fluorescence.NK cell surface thiols group was labeled and quantified by maleimide staining.Furthermore,PX-12 and Torin-1 were used to inhibit Trx and m TOR to validate the importance of thioredoxin system in NK cells’resistance to oxidative stress.3.Intracellular ROS level,NK cell penetration and surface thiol group density were compared between non-small cell lung cancer central tissue,peripheral tissue and paired normal adjacent lung tissue.By analyzing the public single cell sequencing data of NSCLC tumor tissue and paired peripheral blood sample,we investigated the changes of oxidoreductase activity related gene in tumor infiltrating NK cells.The prognostic value of NK cell signature and IL15 gene expression was evaluated in a NSCLC patient cohort from TCGA-LUAD,which was separated into 2 groups depended on the smoking history.Results:1.IL-15 primed NK cells showed greater ability to kill target tumor cells compared with IL-2 primed NK cells,as well as with higher degranulation and IFNγproduction.After exposure to different concentration of H₂O₂,IL-15 primed NK cells were better at maintaining cytotoxicity,degranulation and IFNγproduction.The increased intracellular ROS of IL-15 primed NK cells was significantly lower than IL-2 primed NK cells.2. GSEA result indicated that genes related to oxidoreductase activity were elevated in IL-15 primed NK cells,especially genes within the thioredoxin system.Through q PCR,Western Blot,flow cytometry and immune fluorescence confocal images,we validated that IL-15 upregulates thioredoxin activity in NK cells by both expression and reduced shuttling of mitochondrial TXNIP.Maleimide staining showed that IL-15 primed NK cells had reduced expression of cell surface thiol density.Inhibition of thioredoxin-1reduces NK cell surface thiol density and reverses IL-15 mediated resistance against oxidative stress.Torin-1 inhibition assay proved that m TOR activation suppressed the expression of TXNIP to sustain thioredoxin activity and cell surface thiol density.3. Analysis of tumor resections from different locations showed that NK cells’ penetration was influenced by the accumulation of oxidative stress in NSCLC tumors,while higher surface thiol density could facilitate NK cells to infiltrate into tumor core.From the single cell sequencing data,we found that the oxidoreductase activities were upregulated in tumor-infiltrating NK cells comparing with peripheral blood NK cells.Through analyzing overall survival（OS）and progression free interval（PFI）of the NSCLC patient cohort,we showed the prognostic values of NK cell signature and IL15gene expression in the patients with smoking history.Conclusion:IL-15 conferred resistance in NK cells against oxidative stress through thioredoxin system within the tumor microenvironment,which provides a new potential therapeutic direction for the clinical translation of NK cell immunotherapy in NSCLC.