Igf-1Promotes The Development And Cytotoxic Activity Of Human Nk Cells

Natural killer (NK) cells represent a distinct lymphocyte subset with a central role in innate immunity. NK cells play key roles in innate and adaptive immune responses during early host defense against invading pathogens and tumors. However, the knowledge about the development and the functions of NK cells is still very limited.Insulin-like growth factor (IGF)-1is one of the main endocrine mediators of growth and development under physiological conditions. In addition to this important function, IGF-1also regulates diverse aspects of T-cell and B-cell function through its interactions with IGF-1R. However, very little is known about the role of IGF-1in NK cell development and function. This project will clarify of the role of IGF-1in human NK cell development and function and explore its mechanism, which may be helpful to present new opportunities for boosting or limiting NK cell cytotoxicity and provide new mechanistic insights into the biology of human NK cells.In this project, we firstly detected the role of IGF-1in human NK cell development based on multiple cytokines (SCF, Flt3-L, IL-15and IGF-1) culture for obtaining a high number of NK cells from human umbilical cord blood CD34+cells. Secondly, we assessed the role ofIGF-1in human uterine dNK cells, which possess reduced lytic activity. In our experiment, we found that the dNK cells had a lower production of IGF-1compared with pNK cells.Given the finding that dNK cells produced less IGF-1and that dNK cells had low lytic activity, we hypothesized that the endogenous IGF-1may be important for NK cell cytotoxicity. To confirm the above hypothesis, we designed and synthesised of IGF-1gene siRNA to transfect human NK cell lines. We detected the mRNA levels of PRF1, GzmB and IFNG in human NK cells transfected with IGF-1siRNA. In addition, we examined the lytic activity of pNK cells treated with IGF-1-neutralizing antibody. Finally, we compared miRNA expression profiling of human uterine decidual NK (dNK) cells, cord blood NK (cNK) cells, peripheral blood NK (pNK) cells, T and NKT cells using globle miRNA microarrays. We constructed and identificated the luciferase reporter vectors with IGF-13’UTR and validated the target gene IGF-1of hsa-miR-483-3p by dual luciferase activity report. To confirm the roles of hsa-miR-483-3p in human NK cell cytotoxicity, we detected the role of miR-483-3p mimic and inhibitor on human NK cells and clarified the mechanism of hsa-miR-483-3p in human NK cells.The methods and results in this project were presented according to the contents above which divided into4parts.1. IGF-1promotes the development of human NK cells.We used a classical culture system as a tool to study the role of IGF-1in human NK cell development.Purified CD34+HSCs from human umbilical cord blood (UCB) were maintained with Flt3-L and SCF, IL-15and IGF-1for up to4weeks. To monitor cell expansion in response to cytokine stimulation, cells were counted weekly. At day28, collect cells, the percentage of CD3-CD56+cells was measured by FACS analysis. NK cell function was evaluated through flow cytometry monitoring of perforin and CD107a expression.The results indicated that a significant increase was observed in the percentages and absolute cell numbers of CD3"CD56+NK cells when cells were cultured in the above multiple cytokines with IGF-1. Moreover, We found that SCF, Flt3-L and IL-15with IGF-1significantly increased perform expression and CD107a release in human CD34+-derived CD56+NK cells compared with that of CD56+effectors derived from the same CD34+population in the presence of SCF, Flt3-L and IL-15alone.2. IGF-1induces increased human NK cytotoxic potential.we assessed the role of IGF-1in human uterine dNK cells, which possess reduced lytic activity. We detected the mRNA levels of PRF1, GzmB and IFNG by QRT-PCR and detected Perfotin and CD107a expression by FACS in dNK cells treated with IGF-1,Moreover,We used51Cr release assay against K562to examine the lytic activity of human NK cells.Our data showed that exogenous IGF-1caused a dose-dependent enhancement in the expression of the cytotoxic effector gene PRF1, Moreover, the incubation of dNK cells with K562cells in an IGF-1-containing medium resulted in a substantial increase in the cytotoxic activity of dNK cells.3. Endogenous IGF-1is critical for the cytotoxic activity of human NK cells.In our experiment, we detected the endogenous IGF-1level of human NK cells by ELISA assay and found that dNK cells produced less IGF-1and that dNK cells displayed reduced lytic activity when compared to pNK cells, we hypothesized that the ability to produce endogenous IGF-1may be important for NK cell cytotoxicity. To confirm the above hypothesis, we performed several experiments. We knocked down IGF-1expression using IGF-1siRNA and assayed the mRNA levels of PRF1, GzmB and IFNG by QRT-PCR in human NK cells.In addition, we assessed the requirement of IGF-1for the induction of cytotoxicity in human primary NK cells with IGF-1-neutralizing antibody using51Cr release assay.IGF-1siRNA assay confirmed that knockdown of IGF-1in human NK cells resulted in the downregulation of PRF1gene expression.51Cr release assay confirmed that the lytic activity of pNK cells was largely reduced when pNK cells were treated with an IGF-1-neutralizing antibody, indicating that human NK cell cytotoxicity is dependent on endogenous IGF-1.4. MiR-483-3p regulates human NK cell cytotoxicity by targeting IGF-1.To uncover the underlying mechanisms controlled IGF-1expression in human NK cells, we initially assessed miRNA expression in human primary NK cells with miRNA microarray. Bioinformatics analysis to screen microRNAs that target IGF-1; use of molecular cloning methods to construct the IGF-13’UTR luciferase reporter and the dualluciferase reporters gene assay to validate the interaction of IGF-1and microRNAs. Finally, we examined the effect of miRNA gain-of-function and loss-of function on the cytotoxic activity of human NK cells.The miRNA microarray results indicated that nine miRNAs (miR-340*, miR-483-3p, miR-130a, miR-199b-5p, miR-199a-3p, miR-210and miR-362-5p) have subset-specific expression. We focused on miR-483-3p because it had the greatest difference in expression in dNK cells versus the other subsets, and more importantly, because it was predicted by TargetScan to target the IGF-1gene. Then we successfully constructed the IGF-13’UTR luciferase reporter vectors; validate the interaction of IGF-1and miR-483-3p by the dual luciferase reporter experiments. Finally, we confirmed that overexpression of miR-483-3p decreased natural killer cell cytotoxicity, whereas inhibition of miR-483-3p has the opposite effect, which is reversible with IGF-1neutralizing antibody.Collectively, in this project, we illustrated that IGF-1is a significant modulator of NK cell development and function and that miR-483-3p plays a critical role in regulating NK cell cytotoxic activity.