Unique Phenotypic Characteristics Of Ovarian NK Cells And Their Role In Maintaining Ovarian Homeostasis

Part Ⅰ Phenotypic characterization of ovarian NK cells based on single cell sequencingBackground:The ovary is the main site of follicle development,ovulation,and sex hormone synthesis and secretion.Its proper functioning depends on the coordinated action of multiple cells,and the local immune microenvironment is essential for maintaining its physiological functions.A growing number of studies have shown that the local immune characteristics of tissue organs differ significantly from those of peripheral immunity.This is due to the presence of unique subpopulations of immune cells and functional molecules in tissue organs,which contribute to the distinctive regional immune properties and ensure the homeostasis maintenance of tissue organ.The unique ovarian environment with high steroid hormone,combined with the complex interaction between oocytes and various somatic cells,may result in a distinctive phenotypic and functional remodeling of ovarian immune cells.However,there is still a gap in our understanding of the ovarian local immune properties and their role in maintaining ovarian function.Natural Killer(NK)cells,as the third major class of lymphocytes,exhibit direct cytotoxic effects by recognizing and eliminating target cells rapidly.Furthermore,they possess immunomodulatory functions by synthesizing and releasing different cytokines to regulate the activity of other immune cells.In recent years,tissue-resident NK cells(trNK)have been discovered in various organs,such as the liver,adipose tissue,kidney,salivary gland,and uterus.These trNK cells reside in the local tissues for extended periods and possess unique functional characteristics.There is a high degree of heterogeneity in the functions of trNK cells in different tissues,and they play essential roles in maintaining specific physiological functions of the organs they reside in.While recent studies have focused on trNK cells at the maternal-fetal interface in the female reproductive system,there is still limited research on the composition,phenotypic characteristics,and functions of NK cell subpopulations in the ovary.In the first part of this study,we conducted single-cell transcriptome sequencing of CD45+immune cells in the mouse ovary to analyze the composition of multiple immune cells and to further characterize the phenotypic profile of ovarian-resident NK cells.Objective:To explore the composition of ovarian immune cell subpopulations,as well as the gene expression profiles and distribution of ovarian NK cells subpopulations through single-cell transcriptome sequencing and flow cytometry analysis.Furthermore,we aim to provide a better understanding of the phenotypic characteristics of ovarian-resident NK cells.Methods:In this study,mouse ovarian tissues were collected and subjected to flow cytometric sorting to enrich for CD45+ immune cells.The enriched cells were then used for 10x Genomics 3’end transcriptome sequencing,followed by Seurat screening of highly variable genes for downgraded clustering and differential gene expression analysis.Flow cytometric analysis was also conducted to confirm the phenotypic characteristics of the cell population at protein level.Additionally,a parabiosis model was employed to verify tissue residency of ovarian CD49a+NK cells.Results:(1)Landscape of mouse ovarian immune cellsThe mouse ovaries were found to contain diverse immune cell types,mainly including T cells,monocytes-macrophages,NK cells,dendritic cells(DCs),granulocytes,mast cells,γδ T cells and Group 2 Innate lymphoid cells(ILC2s).These immune cell types were observed to be highly heterogeneous and could be further classified into multiple subpopulations based on the specific genes they express.Additionally,flow cytometry was used to validate the existence of new cell types,such as resident macrophages,resident NK cells,γδT cells,and ILC2s.(2)Phenotypic characterization of ovarian NK cell subsetsThrough analysis of differentially expressed genes(DEGs)in ovarian NK cells,a subpopulation of NK cells with high expression of Itga1(CD49a)was identified.The residency of these CD49a+NK cells was confirmed by parabiosis models.In addition,GO and KEGG enrichment analysis,as well as Monocle pseudotime analysis,were employed to identify differential expression genes between ovarian-resident NK cells and circulating NK cells.Some of these markers were then validated at the protein level.(3)Exploring the dynamic changes of ovarian CD49a+NK cellsThe analysis of ovarian NK cells at different estrous cycles and ages revealed that the proportion of resident NK cells to circulating NK cells was higher during the preoestrus and oestrus periods,and decreased during the postestrus and dioestrus periods.Quantitative analysis suggested that the increase in the number of circulating NK cells in the late period may be the primary reason for the decrease in the proportion of tissue-resident NK cells.Additionally,with aging,both subpopulations of ovarian NK cells exhibited a decline in numbers,with resident NK cells showing a relatively smaller decrease.This resulted in a higher proportion of resident NK cells in older mice.Conclusion:The ovarian immune cell population was found to be highly heterogeneous,with two subsets of NK cells,including CD49a+NK cells,which exhibited tissue residency and unique phenotypic features.Additionally,ovarian-resident NK cells displayed regular fluctuations in their abundance with changes in estrous cycle and aging.Part Ⅱ The role of NK cells in the maintenance of ovarian homeostasis and ovarian agingBackground:In the first part of this study,we utilized single-cell sequencing technology to identify and validate the resident subpopulation of ovarian NK cells,comprehensively exploring their phenotypic characteristics.We observed a decrease in the number of NK cells in the ovary during aging,highlighting the potentially important role of NK cells in maintaining ovarian homeostasis.Similar tissue-specific NK cells have been identified in various tissues,and they have diverse functions and play a crucial role in maintaining specific physiological functions of various tissues.However,abnormal NK cell functions can lead to the development of various inflammatory diseases.For example,decidua-resident NK cells constitute up to 70%of cells and are involved in vascular remodeling,trophoblast invasion,and immune tolerance at the maternal-fetal interface.Additionally,NK cells in adipose tissue are responsible for maintaining homeostasis by eliminating macrophages.However,researches on the phenotype and function of ovarian NK cells is limited,and the impact of ovarian NK cells on immune homeostasis and their involvement in ovarian inflammatory senescence remain unclear.In this study,we aim to elucidate the effects of NK cell depletion on the normal physiological phenotype of ovaries and investigate the potential role of NK cells in ovarian senescence by establishing an autoimmune POI mouse model.Objective:To investigate the potential role of NK cells in maintaining ovarian homeostasis,we treated mice with anti-NK1.1 antibody to explore whether NK cell deletion caused any changes in ovarian phenotype,and utilized an immune POI model combined with NK cell depletion to examine the NK cell response and their contribution to maintaining ovarian homeostasis.Methods:Mice were treated with anti-NK1.1 antibody to observe changes in hormone levels,follicle numbers and other indicators,combined with common transcriptome sequencing and flow cytometry to detect changes of other immune cell subsets in the ovarian microenvironment;pZP3 immunized mice were used to establish an immune POI model combined with NK cell deletion to observe phenotypic changes related to ovarian inflammation,and flow cytometry was used to detect the response of T cells and NK cells in the ovary.Results:(1)Senescent ovarian phenotypes were observed after depletion of NK cellIn this study,normal mice were subjected to NK cell depletion using anti-NK 1.1 monoclonal antibody.Following the treatment,ovarian-related phenotypes were observed,revealing a significant disturbance in the estrous cycle of the treated mice,as well as fewer follicles at all levels and a notable reduction in serum estradiol levels.Further investigation using cleaved PARP labeling demonstrated a significant increase in granulosa cell apoptosis.Furthermore,sequencing data and flow cytometry were used to identify other immune cells present in the ovary,revealing increased macrophage infiltration and polarization towards the M1 subtype.(2)NK cell responded and inhibited overreaction of inflammatory T cells in immune POIIn this study,an immune POI model was established using pZP3-immunized mice.The mice in the modeling group showed disturbed estrous cycles,reduced follicles at all levels,and significantly increased serum follicle-stimulating hormone levels.Additionally,T cells in the ovaries showed increased PD-1 expression,while ovarian NK cells expressed high levels of PD-L1 at the basal physiological level,and their numbers and CD25 expression increased after immunization.Furthermore,NK cell depletion exacerbated the phenotype of autoimmune oophoritis,indicating that NK cells responded and inhibited T-cell overreaction by binding PD-L1 to PD-1.Conclusion:In vivo depletion of NK cells not only induced ovarian senescence,but also exacerbated the phenotype of immune POI mice,suggesting that NK cells play a crucial role in maintaining ovarian homeostasis.The protective effect of NK cells may be mediated through their interactions with macrophages or T cells.Part Ⅲ Abnormal phenotypes and functions of NK cells in patients with premature ovarian insufficiencyBackground:In the second part of our study,we found that NK cell depletion in vivo may contribute to senescence-related changes in the ovary.Additionally,we discovered that NK cell depletion exacerbates the phenotype of autoimmune ovarian inflammation,indicating a possible link between NK cells and ovarian aging.One of the most prevalent reproductive aging disorders in females is early-onset ovarian insufficiency(POI),which is characterized by abnormal menstruation before the age of 40 and endocrine abnormalities such as increased gonadotropin levels and decreased estrogen.The etiology of POI is multifaceted,with autoimmune abnormalities accounting for 5-30%of cases.For a long time,the diagnosis and understanding of autoimmune POI were often linked to other organ-specific or systemic autoimmune diseases.Abnormal autoantibodies,cellular immune imbalance,and autoimmune diseases can damage the ovary and contribute to the development of POI.The local immune microenvironment in the ovary has been insufficiently studied,particularly regarding the pathological changes that occur.Current research on NK cells and ovarian insufficiency mainly focuses on peripheral blood,leaving many unknowns about the characteristics,phenotypes,and relationships with ovarian reserve of local NK cells in the ovary.Recently,single-cell transcriptome sequencing technology has been utilized to describe subpopulations of immune cells in human ovaries,with NK cells being a vital component.However,the phenotypic traits and functions of their subsets remain inadequately researched.Therefore,this aspect of our study aims to investigate the composition and functional characteristics of human ovary NK cell subpopulations by collecting ovarian tissue and follicular fluids and analyzing various functional molecules related to NK cells.Objective:To characterize the subpopulation composition and phenotypic characteristics of NK cells in human ovaries and to clarify the molecular abnormalities of NK cell function in patients with POI.Methods:Peripheral blood,ovarian tissue,and follicular fluid were obtained from human subjects.Flow cytometry was utilized to compare the population and phenotype of NK cells across various samples.In particular,we collected follicular fluid from women with POI and normal ovarian reserve control women to analyze and compare the phenotypic and functional molecules of NK cells using flow cytometry.Results:(1)Identification of CD56brightCD16-/dim CD49a+NK cells as resident cells in human ovaries.Based on our analysis,we observed distinct NK cell subpopulations in peripheral blood,ovarian tissue,and follicular fluid.Specifically,the CD56brightCD16-subpopulation was found to be more abundant in the local ovarian microenvironment compared to the peripheral blood.Moreover,we identified the presence of CD56brghtcCD16-/dimCD49a+NK-resident NK cells in the ovary expressing other resident-associated molecules such as CXCR6,CD69,and CD 103.In comparison to CD56dimCD16+NK cells,CD56brightCD16-NK cells showed a significant decrease in the expression of cell adhesion molecules CD11b and CD62L as well as transcription factor T-bet,while an increase in the expression of the inhibitory receptor NKG2A,indicating the existence of resident NK cells in human ovaries with phenotypic characteristics similar to mice.(2)Abnormal expression of functional phenotype in NK cells in patients with POITo examine whether there were any changes in the expression of functional molecules of NK cell subpopulations,we collected follicular fluid samples from patients with varying levels of ovarian reserve.Our findings indicated a decrease in the expression of Granzyme B in CD56bright CD16-NK cells in the ovaries of individuals with low ovarian reserve.Additionally,there was an increase in CD57 expression in CD56dimCD16+ NK cells.These results suggest that different subsets of NK cells in the ovaries of patients with POI exhibit abnormal phenotypes.Conclusion:Resident NK cells were also present in human ovaries and have phenotypic characteristics similar to those of mice,and different subsets of ovarian NK cells in POI patients showed abnormal phenotypes.