Induction,Identification And Preliminary Application Of Human Decidual-like NK Cells

During the early stages of pregnancy,the endometrium is transformed into decidual tissue by both estrogen and progesterone,forming a maternal-fetal interface that supports embryo implantation and placental growth as a result of the interaction between the mother and fetus.During the first trimester,CD45+leukocytes account for about 40%of the total number of cells at the maternal-fetal interface,in which up to 70%of all leukocytes in human decidua are natural killer(NK)cells.These human decidual NK cells(dNKs)have distinct functional and phenotypic characteristics,including a CD56brghtCD 16" phenotype and high expression of the tissue-resident marker CD49a.They also have strong secretory function and poor cytotoxicity.The dNK cells can maintain immune tolerance at the fetal-maternal interface,promote decidual vascularization,spiral artery formation and extravillous trophoblast invasion by a noncytotoxic mechanism,enhance fetal growth during the critical early stages of fetal development,and contribute to placenta formation.Abnormalities in NK cells,which include lower cell counts or dysfunction,can negatively affect the balance of the fetal-maternal microenvironment and eventually cause pathological pregnancy.Recurrent spontaneous abortion(RSA)and recurrent implantation failure(RIF)are caused by many factors,some of which are not clear and lack reliable screening indicators and effective treatment.At present,it has become a common pregnancy disease troubling pregnant woman,seriously affecting reproductive health.RSA and RIF are generally considered to have similar pathogenic mechanisms and are related to abnormalities in dNK cells at the maternal-fetal interface.Previous studies have shown that in the decidual tissue of patients with unexplained RSA,CD49a+NK cells are significantly reduced,and the function of secreting growth promoting factors(GPFs)is limited.Adoptive transfer of CD49a+decidual-like NK cells to NK cell-deficient mice,which have fertility disorders,can effectively improve the pregnancy outcome of mice.However,there are no related reports or patents of human decidual-like NK cells in the treatment of pregnancy diseases.Therefore,the purpose of this study was to establish a culture system of humaninduced CD49a+decidual-like NK cells(iNKs)in vitro as an auxiliary means in the treatment of pregnancy diseases such as RSA and RIF,and to provide new ideas and methods for the cure of infertility.The following results are obtained:1.The CD49a+iNK cells with proliferation potential were obtained by induction and expansion in vitro:We successfully established three cell-derived iNK cell culture systems in vitro.CB-iNK cells are derived from cord blood hematopoietic stem cells(HSCs);BM-iNK cells are derived from bone marrow HSCs;and pNK-iNK cells are derived from peripheral blood NK cells(pNKs).The induction depends on a distinct combination of cytokines without feeder cells.The iNK cells have high expression of the tissue-resident marker CD49a and significant proliferation potential.2.The iNK cells have similar gene expression profile to dNK cells:We generated purified fresh pNK cells,dNK cells,CB-iNK cells,BM-iNK cells,and pNK-iNK cells and analyzed them by transcriptome sequencing.Through the differentially expressed gene cluster analysis in five kinds of NK cells,we found that there were similar molecular expression profiles between iNK cells and dNK cells.3.The iNK cells exhibit phenotype signatures similar to those of dNK cells:Through the analysis of transcriptome sequencing data and the detection of cell surface molecules by flow cytometry,we found that CB-iNK cells,BM-iNK cells and pNK-iNK cells expressed the characteristic phenotypic molecules of dNK cells,such as CD9,CD151,and CD103.4.The iNK cells have low cytotoxic capability:To evaluate the safety of the clinical application of iNK cells,we assessed the cytotoxicity of iNK cells.We first performed a gene set enrichment analysis of the five kinds of NK transcriptional profiles.Compared to dNK cells and iNK cells,pNK cells were significantly enriched in the pathway of NK cell-mediated cytotoxicity.The heatmap and flow cytometry results showed that iNK cells and dNK cells downregulated the expression of genes related to the NK cell-mediated cytotoxic signaling pathway,such as FCGR3A(CD 16).The results of the killing experiment showed that the killing efficiency of iNK cells and dNK cells against K562 cells was significantly lower than that against pNK cells.The above results suggest that iNK cells have low cytotoxicity.5.The iNK cells present the functional characterization of dNK cells:The iNK cells are similar to dNK cells and contain a high proportion of CD49a+EOMES+ subsets.Through flow cytometry and immunofluorescence detection,we found that iNK cells and dNK cells secrete GPFs.The analysis of transcriptome sequencing results showed that iNK cells and dNK cells highly expressed angiogenesisrelated genes and tissue-residence related chemokines and chemokine receptors.The above results suggest that the iNK cells present the functional characterization of dNK cells.6.The iNK cells promote fetal growth in mice:Because of the high similarity of dNK function-related molecules between human and mouse species,we transferred iNK cells into NCG mice to verify the physiological function of iNK cells in vivo.Through doppler ultrasound detection of pregnant mice and the measurement of murine fetuses and placentas,we found that adoptive transfer of iNK cells can effectively promote vascular development at the maternal-fetal interface,as well as placental and fetal growth in mice.7.The immune status of menstrual blood NK(MBNK)cells in patients with RSA was abnormal:We detected MBNK cells by flow cytometry and found that the proportion of NK cells in menstrual blood CD45+lymphocytes in patients with RSA was significantly lower than that in normal women.Through the bioinformatics analysis of transcriptome sequencing data,there were significant differences in MBNK cells between RSA patients and normal subjects but not in pNK cells,and there was a high similarity between normal MBNK cells and dNK cells.In RSA patients,the expression of resident molecules and inhibitory receptors in MBNK cells decreased,while the expression of cytotoxicity-related molecules increased,showing a state of immune abnormality,suggesting that the appropriate clinical trials of patients can be selected by detecting MBNK cells.8.Establishment of a clinical research program for autologous decidua-like NK cells:To promote the clinical application of iNK cells,we have established a targeted clinical trial program,including research purpose,research object,research method and technical route,curative effect evaluation,research quality control and quality assurance.In summary,we established three systems for inducing and expanding human CD49a+decidua-like NK cells in vitro.Obtained by a variety of cytokine combinations,iNK cells have the phenotypic and functional characteristics of dNK cells,which can effectively promote fetal growth.On this basis,the detection of MBNK cells in RSA patients provides a reliable subject screening and evaluation system for later clinical trials.Promoting the clinical application of iNK cells will help to solve the pregnancy disorders of women of childbearing age,reduce the economic and mental strength of patients and their families,and promote social harmony and stability.