New use of maternal adoptive transfer of decidual nk cells

Abstract

The present application relates to the use of NK cells, which are decidual-like NK cells, preferably decidual NK cells, in the preparation of a medicament or a kit for promoting the behavioral health of offspring and / or alleviating the behavioral abnormalities of offspring, in the preparation of a medicament or a kit for treating or alleviating maternal NK cell abnormalities or defects, and in the preparation of a medicament or a kit for treating or alleviating autism, learning disabilities, intellectual disabilities or emotional disorders, which are administered by adoptive transfer. The present application proves that adoptive transfer of NK cells to the mother can effectively regulate the maternal immune environment, treat or alleviate maternal NK cell abnormalities or defects, promote the behavioral health of offspring, and treat or alleviate the behavioral abnormalities of offspring, which has a wide application prospect in clinical practice.

Description

Technical Field

[0001] This invention relates to the field of immune cell therapy, specifically to the use of maternal adoptive transfer of decidual NK cells in promoting behavioral health in offspring and / or alleviating behavioral abnormalities in offspring. Background Technology

[0002] Maternal decidual natural killer (NK) cells, as a special type of immune cell, have received widespread attention in recent years due to their unique functions during pregnancy. Initially described as large granular lymphocytes, decidual NK cells can regulate the immune response between the mother and placenta, protecting the embryo from attack by the maternal immune system. Furthermore, decidual NK (dNK) cells also secrete growth factors to promote embryonic development, playing a crucial role in embryo implantation and early development.

[0003] With the increasing prevalence of neurodevelopmental disorders such as autism spectrum disorder and learning disabilities in children, the scientific community is conducting in-depth research into their mechanisms. Studies have shown that neurodevelopmental abnormalities are closely related to early embryonic development, especially the maternal immune status, hormone levels, and uterine microenvironment during early pregnancy, which have a significant impact on fetal neurodevelopment. The uterine microenvironment, as the "first environment" for fetal development, directly determines embryonic development and the formation of the nervous system through its immune status, nutrient supply, and hormone levels.

[0004] Immune abnormalities, particularly overactivation of the maternal immune response or lack of immune tolerance, may lead to abnormalities in fetal neurodevelopment, thereby increasing the risk of behavioral and learning disabilities. Summary of the Invention

[0005] In view of the existing technological background, this invention proposes to utilize the characteristics of maternal decidual NK cells to alleviate behavioral abnormalities in offspring and explore their application in neurodevelopmental disorders.

[0006] Our research indicates that dysfunction of maternal decidual NK cells may affect fetal cerebral cortex development and neural connectivity formation, thereby triggering a range of behavioral and cognitive abnormalities. This finding suggests that decidual NK cells not only play a crucial role in placental immune protection but may also exert a potential regulatory role in the development of the fetal nervous system.

[0007] Therefore, studying the role of decidual NK cells in immune regulation and their potential impact on neurodevelopment is of significant clinical importance. Exploring their immunomodulatory function during embryonic development can provide new treatment strategies for the early screening, intervention, and prevention of neurodevelopmental abnormalities such as autism and learning disabilities, offering strong theoretical support and targeted therapy directions for clinical practice.

[0008] This invention combines the application of immune cell therapy and neurobehavioral intervention, especially in the influence and regulation of the next generation's neurobehavioral behavior through maternal immune cells (decidual NK cells), providing a new technical solution for maternal and infant health and the prevention and treatment of diseases related to neurodevelopmental abnormalities.

[0009] One objective of this invention is to discover that maternal NK cells participate in fetal brain development and promote the behavioral health of offspring, thereby providing early warning screening targets for the behavioral health of offspring.

[0010] Another objective of this invention is to discover that maternal adoptive transfer of decidual NK cells helps alleviate offspring behavioral abnormalities caused by NK cell abnormalities or defects, including those caused by autism, learning disability (LD), intellectual disability (ID), or affective disorder or mood disorder.

[0011] In this invention, "adoptive transfer" and "adoptive transfer" can be used interchangeably. Adoptive cell transfer (ACT) is an immunotherapy method that involves reinfusing specific immune cells (such as T cells or NK cells) that have been isolated, purified, induced to expand, etc., into the patient to supplement their corresponding deficiencies or insufficient responses. It is currently mainly used to treat diseases such as tumors and viral infections.

[0012] In this invention, "adoption transfer of the mother" refers to the transfer of the mother through adoption.

[0013] This invention provides a method for regulating the maternal uterine immune environment by adoptive transfer of decidual NK cells from the mother, thereby alleviating behavioral abnormalities in offspring. Specific implementation methods include:

[0014] 1) Establish a pregnant mouse model in which NK cells are eliminated in early pregnancy to demonstrate the important role of maternal NK cells in the healthy formation of behavioral phenotypes such as learning ability and emotional control in offspring.

[0015] 2) Establish a pregnancy model in which decidual NK cells are adopted into NK cell-deficient mice to demonstrate the alleviating effect of maternal NK cells on learning disabilities and autistic phenotypes in offspring.

[0016] Specifically, the present invention provides the following embodiments:

[0017] 1. Use of NK cells in the preparation of a drug or kit for promoting behavioral health in offspring of a subject and / or alleviating behavioral abnormalities in offspring, wherein the NK cells are preferably decidual-like NK cells, more preferably decidual NK cells, and the subject is a pregnant subject.

[0018] 2. Use of NK cells in the preparation of drugs or kits for treating or alleviating maternal NK cell abnormalities or defects, wherein the NK cells are preferably decidual-like NK cells, more preferably decidual NK cells.

[0019] 3. Use of NK cells in the preparation of drugs or kits for the prevention or relief of autism, learning disabilities, intellectual disabilities or mood disorders in the offspring of a subject, wherein the NK cells are preferably decidual-like NK cells, more preferably decidual NK cells, and the subject is a pregnant subject.

[0020] 4. Use of reagents for detecting NK cells in the preparation of drugs or kits for diagnosing autism, learning disabilities, intellectual disabilities, or mood disorders in the offspring of a subject, wherein the NK cells are preferably decidual-like NK cells, more preferably peripheral blood NK cells, and the subject is a pregnant subject; the reagents for detecting NK cells include reagents conventionally used in the prior art for detecting the number or abundance of NK cells, such as reagents for detecting NK cell molecular marker genes, which include primers for amplifying the NK cell molecular marker genes and real-time quantitative PCR reagents, wherein the NK cell molecular marker genes include NCAM1, NCR1, NKG7, KIR2DL1, and GNLY genes.

[0021] 5. A pharmaceutical composition suitable for adoptive transfer, the pharmaceutical composition comprising NK cells, preferably decidual-like NK cells, more preferably decidual NK cells.

[0022] 6. The pharmaceutical composition of item 5, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0023] 7. Use of the pharmaceutical composition described in item 5 or 6 in the preparation of a drug or kit for promoting behavioral health in offspring and / or alleviating behavioral abnormalities in offspring, a drug or kit for treating or alleviating maternal NK cell abnormalities or defects, or a drug or kit for preventing or alleviating autism, learning disabilities, intellectual disabilities, or mood disorders in offspring.

[0024] 8. The use of any one of items 1-4 and 7, or the pharmaceutical composition of item 5 or 6, wherein the pharmaceutical, kit, or pharmaceutical composition is in a form suitable for adoption.

[0025] 9. The use described in item 1 or 7, wherein the offspring behavioral abnormality is caused by maternal NK cell abnormality or defect.

[0026] 10. The use described in Item 1 or 7, wherein the offspring behavioral abnormality refers to abnormal offspring behavioral patterns caused by neurological diseases such as autism spectrum disorder, learning disability, intellectual disability, or emotional disorders, manifested as difficulties in social interaction, learning difficulties, limited cognitive function, or emotional regulation disorders; wherein the offspring behavioral health refers to the offspring's behavioral patterns, learning and memory abilities, emotional management abilities, and social abilities at each developmental stage being consistent with the characteristics of their physiological and psychological development stages and in line with the expected standards of the social environment.

[0027] Specifically, the decidual-like NK cells include decidual NK cells or peripheral blood-derived NK cells cultured in vitro. The preparation and culture method for the in vitro cultured peripheral blood-derived NK cells is as follows: peripheral blood is collected from the patient, and peripheral blood mononuclear cells are separated using density gradient centrifugation. Then, peripheral blood natural killer (pbNK) cells are separated using magnetic bead negative sorting. The separated pbNK cells are cultured in vitro for 7 days to induce CD49a+ tissue-resident NK cell induction. The CD49a+ tissue-resident NK cell induction conditions are: transforming growth factor-β1 (TGF-β1), human chorionic gonadotropin (hCG), and interleukin-15 (IL-15). The final concentrations of TGF-β1, hCG, and IL-15 are 5-10 ng / ml, 10-20 IU / ml, and 10-20 ng / ml, respectively. TGF-β1 (PeproTech, catalog number: AF-100-21C), hCG (Livzon Pharmaceutical Group Co., Ltd., National Drug Approval Number: H44020673), and IL-15 (IL-15 (PeproTech, catalog number: AF-200-15)) were simultaneously added to SCGM medium supplemented with SPS Serum Protein Substitute on day 0 of pNK cell culture. On day 4, half the medium was replaced. The medium used was serum-free SCGM medium (CellGenix, catalog number: 20802-0500). Culture conditions were: constant temperature 37℃, CO2 concentration 5%, relative humidity 95%, and cell culture medium pH 7.2-7.4.

[0028] When used for diagnostic purposes, the NK cells may be peripheral blood NK cells.

[0029] Decidual NK cells are NK cells derived from decidual tissue. They have unique phenotypes and functions, and exhibit tissue residency characteristics.

[0030] This invention provides an innovative screening and immune intervention method that modulates the maternal immune environment through adoptive transfer of decidual NK cells, thereby alleviating behavioral abnormalities in offspring (such as learning disabilities and autism). Specific advantages and effects include:

[0031] 1) Novel Screening and Prevention Strategies: By establishing mouse models of NK cell clearance and NK cell deficiency in early pregnancy, this invention reveals for the first time the crucial role of maternal NK cells in fetal neural development and validates the correlation between low maternal NK cell counts and behavioral abnormalities in offspring in a newborn cohort. This provides a novel theoretical basis for early screening and immune intervention for fetal neural development disorders.

[0032] 2) Precise immune intervention: Adoptive transfer of decidual NK cells can effectively regulate the maternal immune environment and alleviate behavioral abnormalities in offspring. The transferred decidual NK cells mainly home to the maternal uterus; based on the correlation between maternal NK cell deficiency and behavioral abnormalities in offspring, adoptive transfer of maternal decidual NK cells (or related cell products) can restore the number and function of maternal NK cells, thereby achieving effective intervention for behavioral abnormalities in offspring.

[0033] 3) Broad Clinical Application Prospects: By screening for the molecular marker set of maternal NK cells, the risk of behavioral abnormalities in offspring can be predicted in advance. Preconception adoptive transfer of decidual NK cells (or related cell products) can also serve as a preventive treatment, further reducing the occurrence of fetal neurodevelopmental abnormalities. This technology can not only be applied to early screening and immune intervention in high-risk pregnancies, but also provide personalized immunotherapy plans for high-risk pregnant women, reducing the risk of fetal neurodevelopmental abnormalities and improving maternal and infant health. Attached Figure Description

[0034] Figure 1 A displays NCG and Rag1. - / - Nfil3 - / - Results and statistics of open field experiments for groups B6 and BALB / c; Figure 1 B displays NCG and Rag1. - / - Nfil3 - / - Results and statistics of the three-box social experiment in groups B6 and BALB / c; Figure 1 C displays NCG and Rag1. - / - Nfil3 - / - Results and statistics of the modified Y-maze experiment for groups B6 and BALB / c; Figure 1 D displays NCG and Rag1. - / - Nfil3 - / - Results and statistics of rotator experiments in groups B6 and BALB / c; NCG and Rag1 in the figure. - / - Nfil3 - / -B6 and BALB / c represent offspring of NCG mice and Rag1 mice, respectively. - / - Mouse offspring, Nfil3 - / - Mouse offspring, B6 mouse offspring, BALB / c mouse offspring.

[0035] Figure 2 A shows the expression of molecular marker genes (NCAM1, NCR1, NKG7, KIR2DL1 and GNLY) in peripheral NK cells of mothers of children in the behavioral health group, learning disability group and emotion control disorder group during pregnancy; Figure 2 B shows the expression of the T-cell marker gene CD3e and the B-cell marker gene CD20 during pregnancy in the mothers of children in the behavioral health group, learning disability group, and emotion control disorder group. Figure 2 C shows the lymphocyte and white blood cell counts of the mothers of children in the behavioral health group, learning disability group, and emotion control disorder group; Figure 2 D shows the ROC curve analysis of children in the learning disability group and the emotion control disorder group.

[0036] Figure 3 A shows Nfil3 - / - Group, Nfil3 + / + Groups and Nfil3 - / - + Results and statistics of open field experiments by the dNK group; Figure 3 B shows Nfil3 - / - Group (A), Nfil3 - / - + dNK group (B) and Nfil3 + / + Results and statistics of the three-box social experiment in group (C); Figure 3 C shows Nfil3 - / - Group, Nfil3 + / + Groups and Nfil3 - / - + Results and statistics of the improved Y-maze experiment by the dNK group; Figure 3 D shows Nfil3 - / - Group, Nfil3 + / + Groups and Nfil3 - / - + Results and statistics of the rotator experiment in the dNK group; including Nfil3 - / - Indicates the offspring of mice with NK cell deficiency; Nfil3 + / + Nfil3 - / - Offspring of wild-type mice from the same littermate; Nfil3 - / - + dNK indicates Nfil3 - / - Offspring of mice (NK cell-deficient mice that were adopted during pregnancy and received decidual NK cells derived from wild-type B6 mice). Detailed Implementation

[0037] The following examples will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. Unless otherwise specified, the experimental methods in the examples employ conventional techniques in the art, and the experimental reagents are all commercially available products.

[0038] Example 1: Offspring of mice with maternal NK cell deficiency exhibit autistic-like and learning disability phenotypes.

[0039] To investigate the correlation between maternal immune cells and offspring behavioral health, we collected offspring mice from five mouse pregnancy models, including NCG mice (with combined T cell, B cell, and NK cell deficiency), Rag1 mice, and others. - / - Mice (T cell and B cell deficient), Nfil3 - / - Mice included (NK cell deficient), C57BL / 6 (B6) mice, and BALB / c mice; B6 and BALB / c mice served as control mice (without T cell, B cell, or NK cell deficiencies); all mice were purchased from Nanjing Jicui Pharmaceutical Co., Ltd., with BALB / c mice as the background for NCG mice. - / - Mice and Nfil3 - / - The background mice were C57BL / 6 mice. Eight male mice of each genotype were randomly selected (from three pregnant mice of each genotype) and housed in an SPF (specific pathogen-free, free from major potential infections or conditional pathogens and pathogens that would significantly interfere with scientific experiments) environment for 6 weeks. The following behavioral experiments were then conducted to assess the correlation between offspring behavioral health and maternal immune cells.

[0040] 1. Open field test to assess anxiety and autism-like phenotypes in offspring mice.

[0041] In the experiment, mice were placed individually in a 40 cm × 40 cm open field. Their activity time and trajectory in the central and peripheral areas were recorded for 10 minutes. Anxiety was assessed by the length of time mice spent in the central area and the frequency of entry; mice with higher anxiety levels tended to avoid the central area. During the experiment, mouse behavior was recorded using a video monitoring system, and the data was acquired using the video analysis software EthoVision XT14. The time spent in the central area and the number of times mice entered the central area were calculated, and the behavioral differences between the groups were finally compared through statistical analysis.

[0042] The results showed that, compared with the offspring of the two control mice (B6 and BALB / c), NCG mice and Nfil3 mice... - / - Mouse offspring exhibited significantly reduced frequency of entering the central region of the open field and shorter dwell time there, displaying an anxiety-like phenotype. Figure 1 A).

[0043] 2. Three-box socialization test to assess the socialization and social novelty phenotypes of offspring mice.

[0044] The experiment used a three-box setup, consisting of three interconnected areas: an activity box for free movement of the mice and two social boxes containing social cages. Each box measured 20 cm × 30 cm × 22 cm, and the boxes were connected by adjustable doors to facilitate control of the mice's activity area. The experiment was divided into three phases: the first phase was an adaptation period, during which the mice moved freely in the activity box (middle); the second phase was a socialization test, during which the contact time between the mice and the cage containing a littermate mouse (left, M1) and an empty cage (right, E) was recorded; the third phase was a social novelty test, during which the mice were placed in the activity box, and a novel mouse (the one they had not encountered before, M2) was placed in the social cage on the right, and the contact time between the mice and the novel mouse and littermates was recorded for 12 minutes. During the experiment, the mice's behavior was recorded using a video monitoring system, and their interest in social novelty and the frequency and duration of social interactions were analyzed. All data were extracted using the behavioral analysis software EthoVision XT14, and statistical methods were used to compare and analyze differences.

[0045] The results showed that B6 mice, BALB / c mice, and Rag1 mice... - / - Offspring of mice showed more interest in unfamiliar littermates than in an empty cage, and more interest in novel mice than unfamiliar littermates. NCG mice and Nfil3 mice... - / - In the second stage, there was no significant difference in the contact time between the offspring and the left cage containing M1 mice with littermates of unfamiliar mice (left) and the empty cage E (right). Similarly, in the third stage, there was no significant difference in the contact time between the offspring and the M1 cage containing littermates of unfamiliar mice (left) and the M2 novel mouse (right). This indicates that NK cell-deficient mice (NCG mice and Nfil3 mice) - / - The socialization time and social novelty time of offspring from mice were significantly lower than those from NK cell non-deficient mice (B6 mice, BALB / c mice, and Rag1 mice). - / - (mice) progeny, indicating NK cell-deficient mice (NCG mice and Nfil3) - / - Mice) offspring exhibited poor social phenotypes such as depression. Figure 1 B).

[0046] 3. A modified Y-maze was used to assess the learning and memory abilities of offspring mice.

[0047] The Y-maze consists of three interconnected arms, typically of equal length, each measuring 30 cm × 15 cm × 15 cm in a Y-shape. The experiment was divided into two phases: In the training phase, one arm was closed, allowing mice to freely explore the remaining two open arms for 6 minutes. In the memory test phase, the closed arm was reopened as a new arm, and the mouse was placed back in the starting position and allowed to explore freely. The number of times the mouse entered the new arm and the duration of its stay were recorded. The mouse's exploration of the new arm reflects its spatial memory and learning ability. During the experiment, the mouse's activity was recorded using a video monitoring system. All experimental data were collected using the EthoVision XT14 video analysis system. Finally, statistical methods were used to compare the proportion of entries into the new arm to the total number of entries into the old and new arms, the distance traveled, and the time spent in each arm.

[0048] The results showed that, compared with B6 mice, BALB / c mice and Rag1 mice... - / - Compared to the offspring of mice, NCG mice and Nfil3 mice - / - The number of mouse offspring entering the new arm and the residence time in the new arm were significantly reduced, indicating that NK cell-deficient mice (NCG mice and Nfil3 mice) - / - Mice offspring exhibited a poor spatial learning and memory phenotype. Figure 1 C).

[0049] 4. The rotarod test was used to assess the learning and memory abilities of offspring mice.

[0050] The rotundimeter device (purchased from Beijing Zhongshidichuang Technology Development Co., Ltd.) consists of an adjustable-speed circular rotundimeter and a support. The rotundimeter is 3 cm in diameter and 30 cm in length, mounted on a horizontal platform, and its rotation speed is adjustable. The experiment was divided into two phases: In the training phase, mice were initially placed on the rotundimeter and spun, and the time it took for the mice to maintain balance was recorded. The training period lasted three times, each lasting 15 minutes, with a 30-minute interval between each training session. The memory test phase was usually conducted 24 hours after training. The mice were placed on the rotundimeter again, and the rotation speed of the rotundimeter upon fall and the time taken to fall at a speed of 15 revolutions per second were recorded to estimate their learning and memory abilities. All data were recorded using the mouse rotundimeter, and statistical methods were used to compare the performance differences of offspring mice in each group.

[0051] The results showed that NK cell-deficient mice (NCG mice and Nfil3 mice) - / - The drop speed of offspring from mice was significantly lower than that from NK cell-free mice (B6 mice, BALB / c mice, and Rag1 mice). - / - Mice); under a fixed rotation speed mode of 15 revolutions per second, NCG mice and Nfil3 - / - Shorter progeny retention time in mice indicates NK cell deficiency in mice (NCG mice and Nfil3 mice). - / -Mice offspring exhibited a phenotype of poor motor learning and memory abilities. Figure 1 D).

[0052] Therefore, offspring of mice with maternal NK cell deficiency exhibit phenotypes of learning ability and emotional control impairment, indicating an association between maternal NK cell deficiency and behavioral abnormalities in offspring, particularly autism and learning impairment.

[0053] Example 2: Decreased maternal NK cell molecular markers are associated with intellectual disability and mood disorders in offspring.

[0054] To confirm the relationship between maternal NK cells and offspring behavioral health in a population, we analyzed immune cell molecular marker genes in peripheral blood mononuclear cell samples collected and preserved during pregnancy from a prospective cohort of newborn population quality in Hefei (Biomedical ethics approval number: no. 2015002). Based on the assessment results of the subjects' children using the ASQ-3 (Ages and Stages Questionnaires, Third Edition, a widely used child development screening tool designed to assess the abilities of infants and preschool children in multiple developmental domains. It is a standardized questionnaire primarily used to identify potential developmental delays or disorders in children during their developmental stages, thereby providing early intervention and support, and has been widely used internationally in clinical, educational, and research fields.) maternal samples were divided into three groups: 60 healthy controls (no abnormalities in ASQ-3 scores), 37 cases of emotional disorders (scores not meeting the standards in the "Personal and Social Domain" or "Communication" domain), and 30 cases of learning disabilities (scores not meeting the standards in the "Problem Solving" domain of the ASQ-3). Quantitative real-time PCR (qPCR) was used to analyze the expression of NK cell molecular marker genes (NCAM1, NCR1, NKG7, KIR2DL1, and GNLY) and T cell marker gene CD3e and B cell marker gene CD20 (MS4A1) in samples, reflecting the differences in the number of NK cells, T cells, and B cells in maternal mononuclear cells. Gene expression analysis was performed using a Roche 96-well qPCR instrument, and real-time quantification was performed using SYBR Green PCR reagent (Ag11701) from Accurate Biology. Each reaction system had a total volume of 20 µL, including 10 µL of SYBR Green PCR Master Mix, 0.5 µM of forward and reverse primers, 1 µL of cDNA template, and an appropriate amount of RNase-free water. The reaction program was set as follows: 95℃ pre-denaturation for 5 minutes, followed by 40 cycles of amplification under the following conditions: 95℃ denaturation for 15 seconds, 60℃ annealing for 30 seconds, and 72℃ extension for 30 seconds. Fluorescence signals were measured in real time at the end of each amplification cycle, and the expression levels of each gene were quantified by analyzing CT values. Blank controls were included in all experiments to ensure the accuracy and reliability of the data. Primer sequences are as follows:

[0055] NCAM1 forward primer: 5'-GCAGTTTCTCTGCAGGTGGA-3'

[0056] NCAM1 reverse primer: 5'-CTCGCCTGTAACCACACACT-3'

[0057] NCR1 forward primer: 5'-CTAGGCCGGCAGAATCTGAG-3'

[0058] NCR1 reverse primer: 5'-AGCTGGTATTCAACAGCCCC-3'

[0059] NKG7 forward primer: 5'-CATCATATCAGGCTACATCCACG-3'

[0060] NKG7 reverse primer: 5'-GCTGCGGTGGTTGAGACAA-3'

[0061] KIR2DL1 forward primer: 5'-TCCTTCATCGCTGGTGCTCCAA-3'

[0062] KIR2DL1 reverse primer: 5'-TGTCACCTCCTGAGGGTCTTGT-3'

[0063] GNLY forward primer: 5'-GTACTACGACCTGGCAAGAGCC-3'

[0064] GNLY reverse primer: 5'-TCAGACAGGTCCTGTAGTCACG-3'

[0065] CD3e forward primer: 5'-GTAGTAAGTCTGCTGGCCTCC-3'

[0066] CD3e reverse primer: 5'-CCCCAAACGCCAACTGATAA-3'

[0067] CD20 (MS4A1) forward primer: 5'-CTGGTCCAAAACCACTCTTCAGG-3'

[0068] CD20 (MS4A1) reverse primer: 5'-GGCAATGTGGAAGAGCCCATTC-3'

[0069] It was found that the expression of molecular marker genes in peripheral NK cells during pregnancy was significantly lower in mothers of children with learning disabilities and emotional control disorders than in the healthy group, while there was no significant difference in the expression of molecular marker genes in T cells or B cells compared with the healthy group. Figure 2 A, B); while there was no difference in lymphocyte and white blood cell counts among the three groups (A, B); Figure 2C). ROC curve analysis of the learning disability group and the healthy group showed that only the NK cell marker gene CD56 showed significant differences between the learning disability group and the healthy group, and between the emotion control disorder group and the healthy group, while there were no significant differences in T cell and B cell marker genes. Figure 2 (D) The NK cell marker gene CD56 showed a sensitivity of 63.33% and a specificity of 100% in identifying mood disorders, and a sensitivity of 51.35% and a specificity of 95% in identifying intellectual disabilities. This indicates that some mothers of children with intellectual disabilities and mood disorders exhibited reduced numbers or decreased expression of molecular markers in their peripheral NK cells during pregnancy. This finding is helpful for clinical screening of factors related to behavioral abnormalities in children and further demonstrates a potential association between maternal NK cell status and behavioral health in offspring in the population.

[0070] Example 3: Adoptive transfer of decidual NK cells from the mother helps treat or alleviate behavioral abnormalities in offspring of mice with maternal NK cell deficiency.

[0071] The method for isolating and culturing mouse decidual NK cells was a magnetic sorting method. First, decidual tissue from wild-type B6 mice at 10.5 days of gestation was collected and minced with scissors. Then, 5 mg / ml collagenase IV was added for digestion, and the digestion reaction lasted for approximately 1 hour to promote tissue dissociation. The digested cell suspension was filtered through a sieve to remove larger tissue fragments, and then cultured overnight in culture medium to remove adherent cells. The next day, NK cells were magnetically labeled and sorted using the Stemcell EasySep™ Mouse NKCell Isolation Kit (catalog number: 19815) mouse magnetic sorting kit, ultimately enriching and purifying decidual NK cells. On days 8.5 and 9.5 of gestation in the three groups of mice, "Nfil3" cells were collected. - / - The "+ dNK cells" group received adoptive transfer of 2×10⁻⁶ cells via tail vein injection. 6 One decidual NK cell was injected into 200 μL of physiological saline, and the other two groups were injected with the same amount of physiological saline.

[0072] To further verify the correlation between maternal NK cells and offspring behavioral health, we supplemented maternal NK cells based on Example 1 and then observed behavioral abnormalities in offspring, thus establishing Nfil3. - / - A mouse model of NK cell deficiency was created by adoptive transfer of wild-type B6 mouse-derived decidual NK cells during pregnancy, labeled as: "Nfil3". - / - + dNK cells. Additionally, "Nfil3" - / - "Nfil3" refers to mice with NK cell deficiency. + / + "for Nfil3" - / - Wild-type mice from the same littermate. This makes "Nfil3"- / - Female mice in the "+ dNK cells" group were mated with wild-type B6 male mice to induce "Nfil3" - / - "Mating female mice in group A with wild-type B6 male mice resulted in "Nfil3" + / + "Group of female mice and Nfil3" - / - Male mice were mated to obtain offspring mice of the same genotype in each group. From these three groups of pregnant mice offspring, 12 male mice were randomly selected from each group and raised normally in an SPF environment for 6 weeks.

[0073] The behavioral health status of the three groups of offspring mice was assessed using the four behavioral experiments described in Example 1. In the open field experiment, it was found that: "Nfil3..." - / - "The offspring of the adoptive transfer group had lower frequency of entry into the central region and shorter residence time, exhibiting an anxiety phenotype; in contrast, the adoptive transfer group of decidual NK cells ("Nfil3")..." - / - + dNK cells) progeny showed a significant increase in two indicators, approaching "Nfil3 + / + "Group (with "Nfil3") + / + (No significant difference between the groups) Figure 3 A, with "Nfil3 - / - (The group served as the control group for statistical analysis). In the three-box social experiment, it was found that: "Nfil3" - / - "The offspring of the adoptive transfer group showed no significant differences in social behavior, whether in the first stage with cages containing mice or empty cages, or in the second stage with new and old mice; in contrast, the adoptive transfer group of decidual NK cells ("Nfil3") showed poor social behavior." - / - The offspring of "+ dNK cells" showed a greater tendency to socialize with caged mice and new mice in both stages, exhibiting similar behavior to "Nfil3". + / + "Similar sociality among offspring (compared to "Nfil3")" + / + (No significant difference between the groups) Figure 3 B). In the modified Y-maze experiment, it was found that: "Nfil3" - / - "The offspring of the adoptive transfer group performed the worst in learning and memorizing the new and old arms; in contrast, the adoptive transfer group of decidual NK cells ("Nfil3")..." - / - The progeny of "+ dNK cells" showed significant enhancements in the number of times they entered new arms, exploration distance, and exploration time, exhibiting similar characteristics to "Nfil3". + / + "Spatial learning and memory abilities similar in offspring groups ( Figure 3 C, with "Nfil3" - / - (The group served as the control group for statistical analysis). In the rotational motion learning and memory experiment, it was found that: "Nfil3" - / - "The offspring of the adoptive transfer group performed the worst in endurance of shaft rotation speed after training; in contrast, the adoptive transfer group of decidual NK cells ("Nfil3")..." - / -The progeny of "+ dNK cells" showed significant enhancements in both rotational speed tolerance and fixed rotational speed tolerance time, exhibiting similar characteristics to "Nfil3". + / + "The offspring of this group have similar motor learning and memory abilities (compared to "Nfil3")" + / + (No significant difference between the groups) Figure 3 D, with "Nfil3" - / - "The control group was used for statistical analysis."

[0074] The above experiments demonstrate that replenishing and restoring the number of maternal NK cells during pregnancy, especially decidual NK cells, is beneficial to the behavioral health of offspring; it also suggests that for pregnancies with abnormal maternal NK cells, adoptive transfer of decidual NK cells can help restore the behavioral health of offspring.

Claims

1. Use of NK cells in the preparation of drugs or kits for promoting behavioral health in offspring of a subject and / or alleviating behavioral abnormalities in offspring, wherein the NK cells are preferably decidual-like NK cells, more preferably decidual NK cells, and the subject is a pregnant subject.

2. Use of NK cells in the preparation of drugs or kits for treating or alleviating maternal NK cell abnormalities or defects, wherein the NK cells are preferably decidual-like NK cells, more preferably decidual NK cells.

3. Use of NK cells in the preparation of drugs or kits for the prevention or relief of autism, learning disabilities, intellectual disabilities or mood disorders in the offspring of a subject, wherein the NK cells are preferably decidual-like NK cells, more preferably decidual NK cells, and the subject is a pregnant subject.

4. Use of reagents for detecting NK cells in the preparation of drugs or kits for diagnosing autism, learning disabilities, intellectual disabilities, or mood disorders in the offspring of a subject, wherein the NK cells are preferably decidual NK cells, more preferably peripheral blood NK cells, and the subject is a pregnant subject.

5. A pharmaceutical composition suitable for adoptive transfer, the pharmaceutical composition comprising NK cells, preferably decidual-like NK cells, more preferably decidual NK cells.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

7. Use of the pharmaceutical composition of claim 5 or 6 in the preparation of a medicament or kit for promoting behavioral health in offspring and / or alleviating behavioral abnormalities in offspring, a medicament or kit for treating or alleviating maternal NK cell abnormalities or defects, or a medicament or kit for preventing or alleviating autism, learning disabilities, intellectual disabilities, or mood disorders in offspring.

8. The use according to any one of claims 1-4 and 7, or the pharmaceutical composition according to claim 5 or 6, wherein the pharmaceutical, kit, or pharmaceutical composition is in a form suitable for adoption.

9. The use as described in claim 1 or 7, wherein the offspring behavioral abnormality is caused by maternal NK cell abnormality or defect.

10. The use as described in claim 1 or 7, wherein the offspring behavioral abnormality refers to abnormal offspring behavioral patterns caused by neurological diseases such as autism spectrum disorder, learning disability, intellectual disability, or emotional disorder, manifested as difficulties in social interaction, learning difficulties, limited cognitive function, or emotional regulation disorders; wherein the offspring behavioral health refers to the offspring's behavioral patterns, learning and memory abilities, emotional management abilities, and social abilities at each developmental stage conforming to the characteristics of their physiological and psychological development stages and consistent with the expected standards of the social environment.

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