Use of a substance for inhibiting polarization of virtual memory T cells in the manufacture of a medicament for the prevention and / or treatment of PMOS

Abstract

The application provides a substance for inhibiting virtual memory T cell (T VM ) polarization in prevention and / or treatment of polyendocrine metabolic ovary syndrome (PMOS) and in preparation of a drug for preventing and / or treating PMOS. The application proves, through a mouse model and a human sample, that abnormal thymus-ovary axis activation is related to the pathogenesis of PMOS, T cells, especially CD8 + T VM cells are the key to pathogenesis, can aggravate PMOS pathology and prolong the course; further through in vitro experiments, it is found that IFN-alpha combined with IL-4 can strongly induce virtual memory T cell polarization, suggesting that the JAK-STAT pathway is involved; finally, using a JAK inhibitor, T VM cell generation can be blocked in a dose-dependent manner in vitro and in vivo, and PMOS pathology can be reduced, revealing the clinical application value of the JAK inhibitor in PMOS.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the use of a substance for inhibiting virtual memory T cell polarization in the preparation of a medicament for the prevention and / or treatment of PMOS, the use of a substance for inhibiting virtual memory T cell polarization in the prevention and / or treatment of PMOS, and a pharmaceutical composition. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is one of the most common endocrine and metabolic disorders affecting women of reproductive age, classically considered a dysfunction of the hypothalamic-pituitary-gonadal (HPG) axis. Gonadotropin dysregulation and excessive ovarian androgen production are core features of ovulatory dysfunction and polycystic ovary morphology. In addition to reproductive abnormalities, PMOS is often accompanied by insulin resistance, metabolic disorders, and chronic low-grade inflammation, highlighting its systemic nature. Despite extensive research on HPG axis dysfunction, it remains unclear whether organs outside the reproductive endocrine axis actively participate in the occurrence and progression of PMOS.

[0003] The thymus is a major site of T cell development and central immune tolerance. Besides shaping the T cell pool, thymic output is dynamically regulated by endocrine and inflammatory signaling, and has a wide-ranging impact on peripheral immune homeostasis. Alterations in thymic structure or function are associated with chronic inflammation and autoimmune diseases. Notably, sex hormones profoundly influence thymic cell composition and T cell development, suggesting that the endocrine abnormalities characteristic of PMOS may disrupt thymic output. Whether thymic remodeling promotes the pathogenesis of PMOS and affects ovarian immune regulation has not been systematically explored.

[0004] Virtual memory cell T (T VM ) is CD8 + A unique subset of T cells can acquire a memory-like phenotype without sensitization to foreign antigens. Mouse T cells VM Cells typically exhibit CD8 + CD44 + CD49dlow, Human T VM Like cells can manifest as CD3 + CD8 + CD45RA + NKG2A + It is highly sensitive to cytokine and chemokine signaling and can rapidly acquire inflammatory and cytotoxic effects. Current technology has not yet revealed how androgen-induced changes in thymic stromal cells drive T cells. VM Cell formation also did not reveal T VM Furthermore, the mechanisms of ovarian homing, myeloid cell recruitment, and disease maintenance have not been demonstrated, nor has it been shown that JAK inhibition can redirect CD8. +T cell differentiation fate continuously improves PMOS. Summary of the Invention

[0005] To address the aforementioned issues, this application discloses an androgen-thymus-T... VM Cell-ovarian inflammation axis: Androgens induce early thymic involution and decreased thymic output through androgen receptor signaling in thymic stromal cells; thymic-derived T cells, especially CD8... + T VM Cells proliferate in the periphery and are recruited to reproductive tissues; ovarian T cells VM Cells form an inflammatory amplification circuit with neutrophils, monocytes / macrophages, and dendritic cells through inflammatory factors and CCL5. JAK inhibition not only reduces T... VM Cell formation can also bring initial CD8 + T cell differentiation trajectory evolves from T cells characterized by cytotoxicity and high CXCR3 expression. VM State-oriented non-T VM This application provides the use of a substance for inhibiting virtual memory T cell polarization in the preparation of drugs for the prevention and / or treatment of PMOS, as well as a corresponding pharmaceutical composition, and provides biomarkers for stratification of applicable populations and evaluation of efficacy.

[0006] The specific technical solution of this application is as follows: 1. Use of a substance for inhibiting virtual memory T cell polarization in the preparation of a medicament for the prevention and / or treatment of polyendocrine metabolic ovarian syndrome (PMOS).

[0007] 2. Use of a substance for inhibiting virtual memory T cell polarization in the prevention and / or treatment of PMOS.

[0008] 3. The use according to item 1 or 2, wherein the substance used to inhibit virtual memory T cell polarization is a JAK inhibitor.

[0009] 4. The use according to item 3, wherein the JAK inhibitor is a JAK1 and / or JAK2 inhibitor.

[0010] 5. The use according to item 4, wherein the JAK inhibitor is selected from one or more of baricitinib, ruxolitinib, momolotinib, paclinib, fenzotinib, deruxolitinib, tofacitinib, perfetinib, utpatinib, abuxitinib, frigidinib and AZD1480 or a pharmaceutically acceptable salt thereof. Preferably, the JAK inhibitor is baricitinib or a pharmaceutically acceptable salt thereof.

[0011] 6. The use according to item 1 or 2, wherein the virtual memory T cell polarization is promoted by thymic degeneration and decreased thymic output, said thymic degeneration and decreased output being caused by androgen excess via androgen receptor signaling of thymic stromal cells.

[0012] 7. A pharmaceutical composition for the prevention and / or treatment of PMOS, comprising a substance for inhibiting virtual memory T cell polarization.

[0013] 8. The pharmaceutical composition according to claim 7, wherein the substance for inhibiting virtual memory T cell polarization is a JAK inhibitor.

[0014] 9. The pharmaceutical composition according to claim 8, wherein the JAK inhibitor is a JAK1 and / or JAK2 inhibitor.

[0015] 10. The pharmaceutical composition according to claim 9, wherein the JAK inhibitor is selected from one or more of baricitinib, ruxolitinib, momolotinib, paclinib, fenzotinib, deruxolitinib, tofacitinib, perfetinib, utpatinib, abuxitinib, frigidinib and AZD1480 or a pharmaceutically acceptable salt thereof. Preferably, the JAK inhibitor is baricitinib or a pharmaceutically acceptable salt thereof.

[0016] 11. The pharmaceutical composition according to any one of claims 7 to 10, wherein the virtual memory T cell polarization is promoted by thymic degeneration and decreased thymic output, said thymic degeneration and decreased output being caused by androgen excess via androgen receptor signaling of thymic stromal cells.

[0017] 12. The pharmaceutical composition according to any one of claims 7 to 11, further comprising a pharmaceutically acceptable carrier for delivering the substance for inhibiting virtual memory T cell polarization.

[0018] 13. A method for preventing and / or treating PMOS, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition described in any one of items 7 to 12.

[0019] 14. A kit for screening drugs that inhibit virtual memory T cell polarization, comprising: IFN-α and IL-4 for inducing virtual memory T cell formation; and for detecting naïve CD8+. + Reagents for T cells and virtual memory T cells.

[0020] 15. The kit according to claim 14, wherein the detection reagent comprises: Antibodies used to identify at least three biomarkers selected from CD3, CD8, CD44, CD49d, CD45RA, NKG2A, CXCR3, and CCL5; or Nucleic acid detection reagent for detecting at least three biomarkers selected from CD3, CD8, CD44, CD49d, CD45RA, NKG2A, CXCR3, and CCL5.

[0021] 16. A method for screening drugs that inhibit virtual memory T cell polarization, comprising the following steps: Provides initial CD8 + The test cells containing T cells; The test cells were divided into at least a control group and a candidate group. The candidate group was contacted with the candidate, and the test cells in each group were cultured under stimulating conditions that could induce the formation of virtual memory T cells. After culture, the proportion of virtual memory T cells in CD8+ was measured in both the control group and the candidate group. + The proportion of the total number of T cells and / or the number of virtual memory T cells; Compared with the control group, the candidate group had a reduced percentage of virtual memory T cells per CD8 group. + The candidate is identified as a drug capable of inhibiting virtual memory T cell polarization when considering the proportion of the total number of T cells and / or the number of virtual memory T cells.

[0022] 17. The method according to claim 16, wherein the stimulation conditions include type I interferon and interleukin-4; preferably, the type I interferon is IFN-α; more preferably, the stimulation conditions further include anti-CD3 antibody and anti-CD28 antibody.

[0023] 18. The method according to item 16 or 17, wherein the test cells are selected from mouse or human cells derived from: naïve CD8 cells. + T cells, containing naïve CD8 + T cells are peripheral blood mononuclear cells containing naïve CD8. + T cells in spleen cells containing naïve CD8 + T cells in lymph nodes, containing naïve CD8 + T cells are thymic-derived cells and T cells differentiated from pluripotent stem cells.

[0024] 19. The method according to any one of claims 16 to 18, wherein the mouse's virtual memory T cells are activated via CD45 + CD8 + CD44 + CD49d^low phenotype recognition; human virtual memory T cells via CD3 + CD8+ CD45RA + NKG2A + Phenotypic recognition.

[0025] 20. The method according to any one of items 16 to 19, wherein, after culture, one or more indicators selected from the following are also detected: CXCR3, CCL5, NKG7, PRF1, IFNG, GZMB, CD44, CD49d, NKG2A, cytokine release, cytotoxicity, migration ability, chemotaxis, myeloid cell recruitment ability, and interaction with target cells or myeloid cells.

[0026] 21. The method according to item 20, wherein a candidate substance capable of further reducing CXCR3 expression in virtual memory T cells and / or reducing the chemotactic response of the virtual memory T cells to CXCL9, CXCL10 or CXCL11 is identified as a drug capable of inhibiting virtual memory T cell polarization.

[0027] 22. The method of claim 20, wherein candidates that can also reduce CCL5 expression or release of virtual memory T cells and / or reduce the ability of said virtual memory T cells to recruit myeloid cells via CCR1 and / or CCR5 are identified as drugs capable of inhibiting virtual memory T cell polarization.

[0028] 23. The method according to any one of items 16 to 22, wherein at least two concentrations of the candidate are set, a dose-response relationship is obtained, and a comprehensive score is calculated based on at least two of the following: inhibition rate of virtual memory T cells, cell viability, functional indicators, and consistency of repeatability experiments.

[0029] 24. The use of the kit described in item 14 or 15 or the method for screening drugs that inhibit virtual memory T cell polarization as described in any one of items 16 to 23 in screening drugs for the preparation of drugs for the prevention and / or treatment of PMOS.

[0030] Beneficial effects of the invention (1) This application establishes for the first time the androgen-thymus-T2 ... VM The causal chain of cell-ovarian inflammation was investigated, and it was demonstrated that thymic degeneration and decreased output mediated by androgen receptor signaling in thymic stromal cells preceded obvious ovarian pathology.

[0031] (2) This application utilizes the principles of thymus deficiency, adaptive immunodeficiency, and CD8+. + Multiple models, including T cell clearance and adoptive transfer, have demonstrated that T cells... VM Cells have pathogenic causal properties, which can increase disease susceptibility, aggravate pathology, and prolong the duration of disease after androgen withdrawal.

[0032] (3) This application demonstrates that baricitinib does not only produce transient broad-spectrum immunosuppression, but also limits the initial CD8. + T cells turn to T cells VM The drug can differentiate the state, reduce cytotoxicity and CXCR3 expression and redirect its differentiation trajectory, and the protective effect can be maintained after drug withdrawal, which constitutes an unexpected technical effect.

[0033] (4) This application discloses ovarian T VM Cells form an inflammatory amplification circuit with CCL5 and CCR1 / CCR5-associated myeloid cells, and T cells were found to be involved. VM Cells highly express CXCR3, while PMOS ovaries upregulate CXCL9, CXCL10, and CXCL11, providing mechanistic support for reducing ovarian homing and amplifying inflammation.

[0034] (5) This application found that the initial T cell sjTREC of PMOS patients was reduced in human samples, and established human peripheral blood CD3 + CD8 + CD45RA + NKG2A + T VM The cell-like detection method can be combined with DHEA-S to screen PMOS subtypes that are more likely to benefit from immune-targeted therapy. Attached Figure Description

[0035] Figure 1 The PMOS study showed that AR signals in the thymic stroma drive thymic degeneration and impair thymic output, where: a. Principal component analysis (PCA) was performed on covariant genes in a specified tissue based on batch RNA-seq data collected on day 21 after DHEA treatment. b. Heatmap and hierarchical clustering of pairwise correlations of tissue-wide transcriptional alterations in specified organs based on batch RNA-seq; c. Representative morphology and H&E staining of the thymus in control and DHEA-treated mice (left, DHEA 40 mg / kg / day, subcutaneous injection, for 21 days), and quantification of total thymocyte count (right, ***). P = 0.0001, n = 5); d. Representative flow cytometry images (left) and quantification (right) of thymocytes gated at CD4 and CD8 (***) P (SP-CD8) = 0.0002, *** P (SP-CD4) = 0.0007, * P = 0.0468); e. Peripheral blood naive T cells (CD3+) of healthy controls and PMOS patients+ CD8 + CD45RA + NKG2A - The relative abundance of the signal-connecting T-cell receptor cleavage loop (sjTREC) in *** P = 0.0001, n = 5); f. Rag1 - / - Relative abundance of sjTRECs in circulating naive T cells of mice in PBMCs, wild-type control group, and DHEA-treated group (**) P = 0.0011, n = 4); g. Representative morphology of the thymus in mice from the control group, the DHEA-treated group (DHEA 40 mg / kg / day, subcutaneous injection, for 14 days), and the DHEA-treated group receiving flutamide (Flu, 30 mg / kg / day, intraperitoneal injection, for 14 days) (left), and quantitative data on the total number of thymocytes (right, *). P (Ctrl) = 0.0103, * P (Flu+DHEA) = 0.0329, n = 3); h. Representative flow cytometry image of thymocytes (left) and quantification of thymocytes gated at CD4 and CD8 (right) (*) P (DP Ctrl) = 0.0177, * P (DP Flu + DHEA) = 0.0219, * P (CD4 Ctrl) = 0.0263, * P (CD4 Flu+DHEA) = 0.0365); i. C57BL / 6 mice were given intrathymic injections of AAV9-shAR or AAV9-scramble (1×10⁻⁶). 11 A schematic diagram of the experiment involving DHEA treatment (DHEA 20 mg / kg / day, subcutaneous injection) 2 weeks later, followed by thymus collection 4 weeks later. n = 4); j. Representative morphology of the thymus (left) and quantitative analysis of total thymocyte count (right, ***) P (DHEA) = 0.0001, *** P (AAV9-scramble) = 0.0002, * P = 0.0103); k. Representative flow cytometry image of thymocytes (left) and quantification of thymocytes gated at CD4 and CD8 (right) (***)P (DP) = 0.0007, ** P (DP Ctrl vs. AAV9-scramble) = 0.0033, ** P (DP AAV9-scramble vs. AAV9-shAR) = 0.0026,* P (DN) = 0.0383,** P (DN Ctrl vs. DHEA) =0.0097,** P (DN AAV9-scramble vs. AAV9-shAR) = 0.0065, *** P (CD4) = 0.0009, ** P (CD4 Ctrl vs. DHEA) = 0.0040, ** P (CD4 AAV9-scramble vs. AAV9-shAR) = 0.0064, ** P (CD8 Ctrl vs. DHEA) = 0.0012, ** P (CD8 AAV9-scramble vs. AAV9-shAR) =0.0013,** P (CD8 Ctrl vs. AAV9-scramble-DHEA) = 0.0040); Each point represents a mouse; data are expressed as mean ± standard error; statistical significance is determined by two-tailed unpaired Student's... t - The data were evaluated using Tukey's multiple comparison test after tests (c, d), one-way ANOVA (eh), and two-way ANOVA (j, k); the data represent the data from three independent experiments. P A value <0.05 is considered statistically significant (****) P <0.0001; ns, not significant).

[0036] Figure 2 This indicates that thymus-derived T cells are essential for DHEA-induced PMOS, among which: a. Three-week-old female BALB / c wild-type and Foxn1 - / - Schematic diagram of DHEA treatment and analysis in mice (DHEA 40 mg / kg / day, subcutaneous injection, for a total of 21 days). n = 5); b. Representative estrous cycle analysis (left) and quantification of estrous cycles (right, **) P= 0.0021); where P represents proestrus; E represents estrus; M / D represents metestrus / diestrus; c. Serum testosterone levels; d. Representative H&E staining of the ovary (left) and quantitative analysis of the corpus luteum and cystic follicles (right); where the black arrow indicates the corpus luteum; scale bar: 500 μm; e. In the ovary Il1b (**) P = 0.0030), Il6 (*) P = 0.0409) and Tnf (**) P The relative mRNA expression was 0.0094. f. Weight measured at baseline (day 0) and end point (day 21) (**) P = 0.0100); g. Quantitative analysis of the glucose tolerance test (GTT) curve (left) and area under the curve (AUC) (right, *) P = 0.0332); h. C57BL / 6 wild type and Rag1 - / - Schematic diagram of DHEA treatment and analysis in mice (DHEA 40 mg / kg / day, subcutaneous injection, for a total of 21 days). n = 5); i. Control group and DHEA treatment group Rag1 - / - Representative image of the mouse thymus; j. Control group and DHEA treatment group Rag1 - / - Representative H&E staining of mouse thymus; scale bar: 500 μm; k. In the thymus Foxn1 (***) P = 0.0005), Aire (**) P (Uterus) = 0.0002,** P The relative mRNA level (DHEA = 0.0007); l. Representative estrous cycle analysis (left) and quantification of estrous cycles (right, *) P = 0.0240, ** P = 0.0033, n = 4); m. The serum testosterone level of the specified mouse (**) P = 0.0032, *** P = 0.0008); n. Representative H&E staining of the ovary (left) and corpus luteum (***) P = 0.0009) and cystic follicles (*** P = 0.0004) Quantitative (right); where the black arrow indicates the corpus luteum; Scale bar: 500 μm; o. ovaries Il1b (**) P = 0.0037), Il6 (**) P = 0.0062) and Tnf (***) P The relative mRNA level was 0.0007. p. Weight measured at baseline (day 0) and end point (day 21) (*) P = 0.0456); q. GTT curve (left) and AUC quantification (right, **) P = 0.0028); Each point represents one mouse; data are expressed as mean ± standard error; statistical significance was assessed by Tukey multiple comparison test after one-way ANOVA (k) and two-way ANOVA (bg, lq); the data are representative of three independent experiments. P A value <0.05 is considered statistically significant (****) P <0.0001; ns, not significant).

[0037] Figure 3 Display CD8 + Instead of CD4 + T cells drive PMOS pathology and accumulate in reproductive tissues, including: a. C57BL / 6 mice were treated with DHEA-induced PMOS and CD8+. + / CD4 + Schematic diagram of the experiment on T cell exhaustion; b. Representative flow cytometry images showing CD4 levels in peripheral blood of mice treated with isotype antibodies, control groups, and mice treated with anti-CD4 or anti-CD8 antibodies. + and CD8 + T cell depletion efficiency; c. Representative estrous cycle analysis (left) and quantification of estrous cycles (right, ***) P = 0.0003); d. Serum testosterone levels in specified mice (***) P = 0.0001); e. Representative H&E staining of the ovary (left) and corpus luteum (*) P= 0.0149) and cystic follicles (*** P = 0.0002) Quantitative (right); where the black arrow indicates the luteal body; Scale bar: 500 μm; f. In the ovary Il1b , Il6 (**) P = 0.0082) and Tnf (**) P The relative mRNA level was 0.0014. g. Weight measured at baseline (day 0) and end point (day 21) (*) P (Ctrl) = 0.0127, * P (CD8) = 0.0382); h. GTT curve (left) and AUC quantification (right, **) P = 0.0014); i. Representative immunohistochemical (IHC) staining of CD8 expression in the ovaries of the DHEA-treated group; where red arrows indicate representative CD8. + T cell infiltration; Scale bar: 100 μm; Each dot represents a mouse, and each group n = Six animals were included; data are expressed as mean ± standard error; statistical significance was assessed by Tukey's multiple comparison test after two-way ANOVA; the data represent three independent experiments. P A value <0.05 is considered statistically significant (****) P <0.0001; ns, not significant).

[0038] Figure 4 Display T VM Cells undergo early systemic expansion in experimental and human PMOS, among which: a. Spleen CD8+ cells of mice in the control group and DHEA-treated group + Uniform manifold approximation and projection (UMAP) plot of T cell subsets (left) and percentages (right); where T VM Represents virtual memory T cells; b. Dot plot, showing the selected gene on CD8 + Scaled expression in T cell subsets; where the dot size represents the percentage of cells expressing each gene; c. CD8+ in the spleen of mice in the DHEA-treated group and the control group + Pathway enrichment analysis of differentially expressed genes among T cells; d. Spleen CD8+ of mice in the control group and DHEA-treated group +T cell subset T cell chemotactic gene characteristic score (top) and T cell cytotoxic gene characteristic score (bottom). e. CD8 in DHEA-treated mice + Pseudo-temporal trajectory analysis of T cells, colored according to pseudo-temporal progression; f. Peripheral blood of mice in the control group and DHEA-treated group (***) P = 0.0008, n = 5) Spleen (***) P = 0.0006, n = 5) Ovaries (**) P = 0.0017, n = 5) and uterus (**) P = 0.0014, n = 3) Medium T VM Representative flow cytometry images and quantification of cells; g. Peripheral blood T on day 10 after DHEA treatment VM Representative flow cytometry images (left) and quantification (right) of cells. P =0.0029, n = 4); h. Patient circulation T VM Representative flow cytometry images (left) and quantification (right) of cells. P = 0.0046, * P =0.0138, n = 10); The data are expressed as mean ± standard error; statistical significance is determined by two-tailed unpaired Student's... t - Evaluation was performed using the Tukey multiple comparison test (h) after one-way ANOVA (f, g) and the data were representative of three independent experiments. P A value <0.05 is considered statistically significant (****) P <0.0001; ns, not significant).

[0039] Figure 5 Display T VM Cells increase PMOS susceptibility through CCL5-dependent myeloid cell recruitment to the ovary, among which: a. Used for sorting T VM and T CM Representative flow cytometry gating strategies for cell populations (left); Schematic diagram of adoptive transfer experiment and DHEA treatment in C57BL / 6 mice (right, DHEA 20 mg / kg / day, subcutaneous injection, for 21 days). n = 5); bc. Representative estrous cycle analysis (b) and quantification of estrous cycles (c, **P = 0.0087); d. Serum testosterone levels (***) P = 0.0004, ** P = 0.0011); e. Representative H&E staining of the ovary (left) and corpus luteum (**) P (Ctrl) = 0.0030, ** P (T CM () = 0.0088) and cystic follicles (** P (Ctrl) = 0.0013, ** P (T CM ( ) = 0.0025) Quantitative (right); where the black arrow indicates the luteal body; Scale bar: 500 μm; f. In the ovary Il1b (***) P (Ctrl) = 0.0002, *** P (T CM ) = 0.0001), Il6 (*) P = 0.0226) and Tnf (***) P (CTRL) = 0.0008, *** P (T CM The relative mRNA level () = 0.0007); g. Weight measured at baseline (day 0) and endpoint (day 21) (**) P (Ctrl) = 0.0028, ** P (T CM =0.0089). h. GTT curve (left) and AUC quantification (right, *) P (Ctrl) = 0.0445, * P (T CM () = 0.0500); i. UMAP plots (left) and percentages (right) of major immune cell populations in the ovaries of mice in the control and DHEA-treated groups; j. Ovarian CD8 in control and DHEA-treated mice + UMAP plot of T cells (left) and percentage (right); k. Intercellular communication network analysis reveals inferred interactions between ovarian immune cell subsets; where the thickness of the lines indicates the number of interactions. l. Involves ovarian CD8 +A dot plot of significant ligand-receptor interactions between T cells and myeloid cell populations; where the size of the dots indicates statistical significance and the intensity of the color indicates the probability of communication. m. Dot plot, showing ovarian CD8 + Expression of chemokine ligands in T cells (left) and expression of chemokine receptors in myeloid cell populations (right); Mono / Mac represents monocytes / macrophages; DC represents dendritic cells; Each point represents one mouse; data are expressed as mean ± standard error; statistical significance was assessed by Tukey's multiple comparison test after one-way ANOVA; the data are representative of three independent experiments. P A value <0.05 is considered statistically significant (****) P <0.0001; ns, not significant).

[0040] Figure 6 This shows that JAK inhibitors limit T... VM Cell differentiation alleviates PMOS, among which: a. DHEA model (40 mg / kg / day, subcutaneous injection, for a total of 21 days), n = 5) Schematic diagram of treatment with baricitinib (30 mg / kg / day, administered by gavage for 21 days; solvent control was ddH2O); b. Peripheral blood T VM Representative flow cytometry images (left) and quantification (right, *) of cells. P (Percentage) = 0.0439, ** P = 0.0010, * P (Cell count) = 0.0146). c. Representative estrous cycle analysis (left) and quantification of estrous cycles (right, ***) P = 0.0001); d. Specify the serum testosterone level in the mice; e. Representative H&E staining of the ovary (left) and corpus luteum (***) P = 0.0004, * P (Ctrl) = 0.0308, * P (JAKi) = 0.0195) and cystic follicles (*** P (Ctrl) = 0.0001, *** P (JAKi) = 0.0005) Quantitative (right); f. Representative flow cytometry images (left) and quantitative data (right) of T cell subsets in the ovary. P = 0.0041, * P =0.0301,n = 3); g. Rag1 - / - Mouse DHEA treatment, initial CD8 + Schematic diagram of T cell adoptive transfer and JAK inhibitor treatment and analysis ( n = 4); h. Representative estrous cycle analysis (left) and quantification of estrous cycles (right, **) P (DHEA vs. DHEA+CD8 + T) = 0.0012, ** P (DHEA+CD8 + T vs. DHEA+CD8 + (T+JAKi) = 0.0012). i. Representative H&E staining of the ovary (left) and corpus luteum (*) P = 0.0272, *** P = 0.0001) and cystic follicles (*** P = 0.0001) Quantitative (right); where the black arrow indicates the corpus luteum; Scale bar: 500 μm; j. Serum testosterone levels (***) P = 0.0005); k. Accept adoption transfer initial CD8 + T cells Rag1 - / - In mice, peripheral blood (*) was compared between treatment with and without a JAK inhibitor (JAKi). P = 0.0485), lymph nodes (** P = 0.0061) and spleen (** P = 0.0082) in T VM Representative flow cytometry images and quantification of cells ( n = 3); The data are expressed as mean ± standard error; statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test (bj) and two-tailed unpaired Student's test. t - Test (k) evaluation; the data are representative of three independent experiments; P A value <0.05 is considered statistically significant (****) P <0.0001; ns, not significant).

[0041] Figure 7 The results show that DHEA induces PMOS-like features accompanied by thymic transcriptional remodeling, in which: a. Schematic diagram of the DHEA-induced PMOS model in three-week-old female C57BL / 6 mice (40 mg / kg / day DHEA, subcutaneous injection, for 21 consecutive days). n = 5); b. Representative estrous cycle analysis (left) and quantitative analysis of the estrous cycle (right, **) P = 0.0012). P, proestrus; E, estrus; M / D, metestrus / diestrus. c. Serum testosterone levels; d. Representative hematoxylin and eosin (H&E) staining of the ovary (left) and quantitative analysis of the corpus luteum and cystic follicles (***) P = 0.0004 (right). The black arrow indicates the luteal body. Scale bar: 500 μm; e. In the ovary Il1b (**) P = 0.0031), Il6 (**) P = 0.0095) and Tnf (*) P The relative mRNA expression was 0.0114 (= 0.0114); f. Representative morphology of the uterus; g. in the uterus Il1b , Il6 (***) P = 0.0001) and Tnf The relative mRNA expression; h. Body weight measured at baseline (day 0) and endpoint (day 21) (***) P = 0.0003).

[0042] i. Quantitative analysis of the glucose tolerance test (GTT) curve (left) and the area under the curve (AUC) (right), ** P = 0.0092); jl. Genes associated with fibrotic remodeling in the ovary (j; Tgfbr2 ,*** P = 0.0010; Pbrm1 , * P =0.0232; Smad3 , * P = 0.0369), inflammation-related genes (k; Tlr3 , *P = 0.0281; Edn1 ,*** P = 0.006; Spon2 , * P = 0.0225) and stress-related pathway genes (l; Blm ,** P = 0.0031; Btg2 , * P = 0.0499; Ngfr ,** P Quantitative PCR analysis (= 0.0056) n = 4); m. GO enrichment analysis of co-regulated genes in the thymus and ovary after DHEA treatment. Enriched pathways were categorized into fibrotic remodeling, inflammatory response, and stress / metabolic-related categories.

[0043] In this context, each point represents a mouse; data are expressed as mean ± sem; statistical significance is determined using two-tailed unpaired Student's score. t - Tests (ce, g, il) and two-way ANOVA were performed; followed by Tukey's multiple comparison test (b, h); the data are representative of three independent experiments; P <0.05 is considered statistically significant (****) P <0.0001; ns, no significance). Figure 8 The AR signal in the thymic stroma was shown to drive early thymic regression in various PMOS models, including: a. Schematic diagram of the PMOS model induced by letrozole (LET) in C57BL / 6 mice (1 mg / kg / day letrozole, administered by gavage for 21 consecutive days). n = 4); b. Representative estrous cycle analysis (left) and quantitative analysis of the estrous cycle (right, **) P = 0.0011, * P =0.0126); c. Serum testosterone levels (*) P = 0.2320); d. Representative H&E staining of the ovary (left) and the corpus luteum (**) P = 0.0021) and cystic follicles (** P Quantitative analysis of (= 0.0069) (right); black arrow indicates corpus luteum; scale bar: 500 μm; e. Quantitative analysis of thymus morphology (left) and total number of thymocytes (right, *)P = 0.032); f. Flow cytometry analysis of representative thymocytes (left) and quantitative analysis of absolute cell count (right, *) P (DP) = 0.0444, ** P = 0.0047, * P (SP-CD4) = 0.0199, * P (DN) = 0.0166). g. Quantitative analysis of thymocyte count (*) P (Day 7) = 0.0325, * P (Day 14) = 0.0345, ** P =0.0066, n = 3); h. Representative morphology of the thymus (upper) and uterus (lower) on days 0, 3, 7, 14, and 21; i. In the ovary Il1b , Il6 and Tnf Relative mRNA expression ( n = 3); j. Intravenous injection (iv) AAV9-shAR (2×10) 11 Representative immunoblotting analysis of AR expression in the liver and prostate after vg / mouse; k. Intrathymic injection of AAV9-shAR (1×10⁻⁶) 11 Representative immunoblotting analysis of AR expression in intact thymic tissue, purified thymic T cells, and prostate tissue after vg / mouse; l. Rag1 - / - Intrathymic delivery of AAV9-shAR (1×10⁻⁶) in mice 11 Representative immunoblotting analysis of AR expression in retrothymic tissue (vg / mouse); m. Intrathymic injection (lanes 2-5, 1×10 11 VG / mouse) or intravenous injection (lanes 6 and 7, 2 × 10) 11 Representative immunoblotting analysis of AR expression in the prostate, thymus, and liver after AAV9-shAR (vg / mouse); In this context, each point represents a mouse; data are expressed as mean ± sem; statistical significance is determined using two-tailed unpaired Student's score. t - Test (cf) and two-way ANOVA (b, g, i), followed by Tukey multiple comparison test; the data are representative of three independent experiments;P <0.05 is considered statistically significant (****) P <0.0001; ns, no significance).

[0044] Figure 9 Display T VM Cells expand in the early stages of PMOS, during which: a. CD8 + A schematic diagram of the experimental design for T-cell single-cell RNA sequencing. b UMAP Display and Virtual Memory CD8 + T(T) VM ) Classical gene expression related to cellular procedures and associated phenotypic markers; color indicates normalized expression level; c. Used to identify T VM Cells (CD45) + CD8 + CD49d lo CD44 + Representative flow cytometry gating strategies; d. Splenic T on day 10 after DHEA treatment VM Representative flow cytometry images of cells (left) and quantitative analysis (right, *) P = 0.0205); e. Peripheral blood T VM Representative flow cytometry plot of cells (left) and quantitative analysis, including cell frequency and absolute cell number (*). P = 0.0182) (right); f. Spleen T VM Representative flow cytometry plot of cells (left) and quantitative analysis, including cell frequency and absolute cell number (*). P = 0.0018) (right); g. Used for identifying T in human peripheral blood VM Cells (CD3) + CD8 + CD45RA + NKG2A + ) and naive T cells (CD3) + CD8 + CD45RA + NKG2A - Representative flow cytometry gating strategies; Data are expressed as mean ± sem; each point represents one mouse, n = 4; statistical significance was determined using two-tailed unpaired Student's Law. t- test; P<0.05 is considered statistically significant (****) P <0.0001; ns, no significance).

[0045] Figure 10 Display T VM Cells prolong PMOS pathology after androgen withdrawal by maintaining ovarian inflammation, in which: a. Schematic diagram of adoptive transfer experiment and DHEA treatment in C57BL / 6 mice (40 mg / kg / day DHEA, subcutaneous injection, for 21 consecutive days). n = 5); b. Representative estrous cycle analysis (left) and quantitative analysis of the estrous cycle (right); c. Representative estrous cycle analysis (left) and quantitative analysis of the estrous cycle (right, ** P = 0.0029); d. Serum testosterone levels in mice shown (**) P = 0.0042, * P = 0.0183); e. Representative H&E staining of the ovary (left) and corpus luteum (***) P (Ctrl) = 0.0002, *** P (T) CM =0.0005) and cystic follicles (** P = 0.0063, * P Quantitative analysis of (= 0.0168) (right); black arrow indicates corpus luteum; scale bar: 500 μm; f. Body weight measured at baseline (day 0) and experimental endpoint (day 21) (*P(Ctrl) = 0.0291, *P(T)) CM = 0.0422); g. In the ovary Il1b (*) P (DHEA) = 0.0120, * P (T) CM = 0.0201) Il6 (***) P = 0.0007, ** P =0.0066) and Tnf (**) P (Ctrl) = 0.0007, ** P (T) CM The relative mRNA expression level was 0.0086. h. GTT curve (left) and AUC quantitative analysis (right, *)P = 0.0230, * P = 0.0074); i. Dot plot showing the expression of classic marker genes used in the major ovarian immune cell populations identified by single-cell RNA sequencing; j. Dot plot showing the expression of representative marker genes used to define CD8+ T cell subsets; k, l. Characteristic density plot (k) and violin plot (l) show immune cells in the ovarian ovaries of control and PMOS mice. Il1b , Il6 and Tnf Distribution of expressions; Each point represents one mouse; data are expressed as mean ± sem; statistical significance was determined using one-way ANOVA followed by Tukey's multiple comparison test; the data are representative of three independent experiments. P <0.05 is considered statistically significant (****) P <0.0001; ns, no significance).

[0046] Figure 11 Shows JAK inhibition limit T VM Cell development and improvement of PMOS pathology, including: a. Representative flow cytometry image (left) and T cells generated in an in vitro experiment after 24 hours. VM Quantitative analysis of cells (right, **) P = 0.0040, *** P = 0.0002, n = 3); b. Representative flow cytometry plot (left) of the dose-dependent effects of baricitinib (0-100 nM, 24 hours) and T cells generated in vitro. VM Quantitative analysis of cells (right, **) P = 0.0035, *** P = 0.0001); c. Representative flow cytometry image (left) and spleen T cells VM Quantitative analysis of cells (right, **) P = 0.0090, *** P =0.0005, n = 5); d. Uterine morphology; e. In the ovary Il1b (*) P (Ctrl) = 0.0163, * P(JAKi) = 0.0058) Il6 (*) P (Ctrl) = 0.0419, * P (JAKi) = 0.0412) and Tnf (***) P = 0.0001, *P (Ctrl) = 0.0125, * P The relative mRNA expression level of (DHEA) = 0.0108; f. Body weight measured at baseline (day 0) and experimental endpoint (day 21) (***) P = 0.0004); g. GTT curve (left) and AUC quantitative analysis (right, *) P (Ctrl) = 0.0376, * P (JAKi = 0.0284); Each point represents one mouse; data are expressed as mean ± sem; statistical significance was determined using one-way ANOVA followed by Tukey's multiple comparison test; the data are representative of three independent experiments. P <0.05 is considered statistically significant (****) P <0.0001; ns, no significance).

[0047] Figure 12 The JAK inhibitor shows that CD8 + T cell differentiation redirection, causing it to deviate from the T cell differentiation process. VM Fate and promotes the resolution of inflammation, among which: a. On the 10th day after adoption transfer Rag1 - / - A schematic diagram of spleen cells isolated from mice, and T cells sorted by flow cytometry. VM Cells and T CM Cells are used for bulk RNA sequencing; b. PCA Display T VM Cells and T CM Cells have different transcriptomic characteristics; c. T VM Cells and T CM Heatmap of differentially expressed genes between cells; the figure shows representative genes related to cytotoxicity, chemotaxis, interferon response, proliferation, and metabolism; d. T VM Functional enrichment analysis of upregulated genes in cells; the figure shows the five pathways with the highest enrichment. e. Cxcr3relative mRNA levels (**) P = 0.0026)( n = 3); f. In the ovary Cxcl9 (*) P = 0.0287), Cxcl10 (**) P = 0.0029) and Cxcl11 (****) P The relative mRNA level <0.0001 ( n = 4); g. T CM Enrichment analysis of upregulated genes in cells using gene ontology (GO) biological processes; the figure shows the biological processes with the highest significant enrichment. h. Schematic diagram of the hypothesis regarding the androgen-driven "thymus-peripheral immunity-ovary" loop in PMOS; Data are expressed as mean ± sem; statistical significance is determined using two-tailed unpaired Student's score. t - Test; the data are representative of three independent experiments; P A value <0.05 is considered statistically significant. Detailed Implementation

[0048] Specific embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0049] On the one hand, this application provides the use of a substance for inhibiting virtual memory T cell polarization in the preparation of a medicament for the prevention and / or treatment of PMOS.

[0050] In this application, "cell polarization" refers to the process by which naive T cells, after receiving specific microenvironmental signals (such as antigens, cytokines, etc.), differentiate into a subset of T cells with specific functional phenotypes and effector modes.

[0051] The "virtual memory T cells" in this application are a type of CD8 cell precursor that differentiates from autoreactive T cells in the thymus or periphery via non-antigen-dependent homeostatic and cytokine signaling. + T cell subsets, whose phenotype and some functions are similar to memory T cells, but lack memory of specific foreign antigens, play a rapid response role in infection immunity and immunopathology.

[0052] The term "androgen-thymus-T" in this application VM The "cell-ovarian inflammation axis" refers to the excessive activation of androgen receptor signaling in thymic stromal cells by androgens, leading to thymic degeneration, decreased thymic output, and peripheral CD8+. + T cell homeostasis remodeling, promoting T VM Cell expansion; T VM Cells are further recruited to the ovary and recruit myeloid cells through inflammatory factors and chemokines, amplifying and maintaining the immunoendocrine regulatory pathways of ovarian inflammation.

[0053] In some implementations, inhibiting virtual memory T cell polarization includes inhibiting naïve CD8. + T cells are directed to virtual memory CD8 + T cell differentiation, or redirection of its differentiation pathway, causing it to deviate from the virtual memory CD8. + T cell fate.

[0054] In some implementations, the substance used to inhibit virtual memory T cell polarization is a JAK inhibitor.

[0055] JAK inhibitors (Janus kinase inhibitors) are a class of small molecule compounds that selectively inhibit the activity of the JAK kinase family. JAK kinases are key initiating elements of the intracellular JAK-STAT signaling pathway, responsible for receiving signals from cytokines (such as IFN-α and IL-4) and growth factors, and phosphorylating downstream STAT proteins, thereby regulating gene transcription. By blocking JAK activity, JAK inhibitors can effectively inhibit various pro-inflammatory and autoimmune-related cytokine signals, and are therefore widely used clinically to treat autoimmune diseases such as rheumatoid arthritis and ulcerative colitis.

[0056] In some embodiments, the JAK inhibitor is a JAK1 and / or JAK2 inhibitor. In some embodiments, the JAK inhibitor is a JAK1 inhibitor. In some embodiments, the JAK inhibitor is a JAK2 inhibitor. In some embodiments, the JAK inhibitor is a dual inhibitor of JAK1 and JAK2.

[0057] In some embodiments, the JAK inhibitor is selected from at least one of baricitinib, ruxolitinib, momelotinib, peficitinib, Fedratinib, deuruxolitinib, tofacitinib, peficitinib, upadacitinib, abrocitinib, filgotinib, and AZD1480.

[0058] In some embodiments, the JAK inhibitor is baricitinib. In some embodiments, the JAK inhibitor is baricitinib or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof.

[0059] In some implementations, the virtual memory T cell polarization is promoted by thymic degeneration and decreased thymic output, which are caused by androgen excess via androgen receptor signaling in thymic stromal cells.

[0060] On the other hand, this application also provides the use of a substance for inhibiting virtual memory T cell polarization in the prevention and / or treatment of PMOS.

[0061] In some implementations, the substance used to inhibit virtual memory T cell polarization is a JAK inhibitor.

[0062] In some embodiments, the JAK inhibitor is a JAK1 and / or JAK2 inhibitor. In some embodiments, the JAK inhibitor is a JAK1 inhibitor. In some embodiments, the JAK inhibitor is a JAK2 inhibitor. In some embodiments, the JAK inhibitor is a dual inhibitor of JAK1 and JAK2.

[0063] In some embodiments, the JAK inhibitor is selected from at least one of baricitinib, ruxolitinib, momolotinib, paktinib, fenzotinib, deruxolitinib, tofacitinib, perfetinib, utpatinib, abuxitinib, frigidinib, and AZD1480.

[0064] In some embodiments, the JAK inhibitor is baricitinib. In some embodiments, the JAK inhibitor is baricitinib or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof.

[0065] In some implementations, the virtual memory T cell polarization is promoted by thymic degeneration and decreased thymic output, which are caused by androgen excess via androgen receptor signaling in thymic stromal cells.

[0066] In some implementation schemes, mouse T VM Cells via CD8 + CD44 + CD49d low Phenotypic identification; Human T VM Like cells via CD3 + CD8 + CD45RA + NKG2A + Phenotypic identification.

[0067] In some implementations, substances used to inhibit virtual memory T cell polarization reduce virtual memory CD8. + The proportion or number of T cells, and / or the expression of one or more of CXCR3, CCL5, NKG7, PRF1, and IFNG is reduced.

[0068] In some implementations, substances used to inhibit virtual memory T cell polarization reduce virtual memory CD8. + T cells are recruited or infiltrated into the ovary or other reproductive tissues.

[0069] In some implementations, substances used to inhibit virtual memory T cell polarization reduce the recruitment or accumulation of one or more of neutrophils, monocytes, macrophages, and dendritic cells in the ovary.

[0070] In some implementations, substances used to inhibit virtual memory T cell polarization improve at least one of the following PMOS-related abnormalities: ovulation disorders, estrus or menstrual cycle abnormalities, hyperandrogenemia, cystic follicle hyperplasia, decreased corpus luteum, ovarian or uterine inflammation, weight gain, insulin resistance, or impaired glucose tolerance.

[0071] In some implementations, substances used to inhibit virtual memory T cell polarization cause initial CD8... + T cell reduction leads to T cells VM State differentiation and increase entry into T CM Set the non-T gate area VM Cells; the non-T cells VM Cellular phase compared to T VM The cells exhibited low cytotoxicity and low CXCR3 expression.

[0072] In another aspect, this application also provides a pharmaceutical composition for the prevention and / or treatment of PMOS, comprising a substance for inhibiting virtual memory T cell polarization.

[0073] In this application, "pharmaceutical composition" refers to a mixture containing one or more substances for inhibiting virtual memory T cell polarization or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers or excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, thereby promoting the absorption of the active ingredient and enabling it to exert its biological activity.

[0074] In some implementations, the substance used to inhibit virtual memory T cell polarization is a JAK inhibitor.

[0075] In some embodiments, the JAK inhibitor is a JAK1 and / or JAK2 inhibitor. In some embodiments, the JAK inhibitor is a JAK1 inhibitor. In some embodiments, the JAK inhibitor is a JAK2 inhibitor. In some embodiments, the JAK inhibitor is a dual inhibitor of JAK1 and JAK2.

[0076] In some embodiments, the JAK inhibitor is selected from one or more of baricitinib, ruxolitinib, momolotinib, paklatinib, fenzotinib, deruxolitinib, tofacitinib, perfetinib, utpatinib, abuxitinib, frigidinib, and AZD1480, or a pharmaceutically acceptable salt thereof.

[0077] In some embodiments, the JAK inhibitor is baricitinib. In some embodiments, the JAK inhibitor is baricitinib or a pharmaceutically acceptable salt thereof.

[0078] In some implementations, the virtual memory T cell polarization is promoted by thymic degeneration and decreased thymic output, which are caused by androgen excess via androgen receptor signaling in thymic stromal cells.

[0079] In some embodiments, the pharmaceutical compositions of this application further comprise a pharmaceutically acceptable carrier for delivering a substance for inhibiting virtual memory T cell polarization. "Pharmaceutically acceptable" means a compound, material, composition, or dosage form suitable for tissue contact with human and animal subjects without excessive toxicity, irritation, allergic reactions, or other problems or complications, and consistent with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to substances that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. "Pharmaceutically acceptable carriers" include, but are not limited to, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, disintegrants, stabilizers, solvents, or emulsifiers.

[0080] On the other hand, this application also provides a method for preventing and / or treating PMOS, comprising administering to a subject a therapeutically effective amount of any of the aforementioned pharmaceutical compositions.

[0081] In this application, "administration" or "giving" refers to methods that enable the delivery of a compound or composition to a desired biological site of action. These methods include, but are not limited to, oral or parenteral administration (including intraventricular, intravenous, subcutaneous, intraperitoneal, intramuscular, and intravascular injection or infusion), local administration, and rectal administration. In particular, injection or oral administration.

[0082] The term "treatment" in this application includes relieving, reducing, or improving a disease or symptom; preventing other symptoms; improving or preventing underlying metabolic factors of symptoms; inhibiting a disease or symptom, for example, preventing the development of a disease or symptom; reducing a disease or symptom; promoting the relief of a disease or symptom; or causing the cessation of symptoms of a disease or symptom; and extends to include prevention. "Treatment" also includes achieving therapeutic and / or preventive benefits. A therapeutic benefit refers to the eradication or improvement of the treated condition. A preventive benefit refers to a patient using the composition to prevent the risk of a certain disease, or taking it when a patient experiences one or more disease-related physiological symptoms, even though the disease has not yet been diagnosed.

[0083] In this application, "prevention" refers to any intervention aimed at reducing the risk or likelihood of a subject contracting, contracting, or developing a disease, condition, or symptom. This includes, but is not limited to, administering the composition of this application to prevent the onset of a disease or condition before the subject develops clinical symptoms of the disease or condition, and administering the composition of this application to delay, block, or reverse the disease progression when the subject has developed one or more prodromal symptoms, abnormal biomarkers, or changes in physiological indicators related to the disease or condition but has not yet met the clinical diagnostic criteria for the disease. It also includes interventions for subjects with known disease risk factors such as genetic susceptibility, family history, or environmental exposure, aimed at delaying the onset of the disease or reducing its severity. Preventive benefit refers to the beneficial clinical effects obtained by the subject through the application of the composition of this invention, such as reduced risk of disease occurrence, delayed onset, or symptom relief, even though the subject has not yet been diagnosed with the disease or symptom.

[0084] In this application, "effective amount" or "therapeutic effective amount" refers to a sufficient dosage of a drug or pharmaceutical agent that provides acceptable side effects while achieving the desired therapeutic effect. The determination of the effective amount varies from person to person, depending on the individual's age and general condition, as well as the specific active substance. In any given case, the appropriate effective amount can be determined by a person skilled in the art based on routine testing.

[0085] In this application, "subject" refers to a human or a non-human animal (e.g., a mammal, bird, reptile, fish, amphibian, or invertebrate). The subject can be, for example, a human, wild animal, domesticated animal, or laboratory animal; examples include: mammals such as humans, non-human primates (chimpanzees, bonobos, gorillas), dogs, cats, rodents (e.g., mice, guinea pigs, rats, hamsters or rabbits, horses, donkeys, cattle, sheep, goats, pigs, camels); birds such as ducks, pigeons, turkeys, geese, or chickens; reptiles such as turtles, snakes, lizards; amphibians such as frogs; fish such as zebrafish; and invertebrates such as worms or insects. More preferably, the subject is a "patient."

[0086] In some implementation schemes, subjects can be assessed based on peripheral blood T... VM Screening can be performed based on one or more of the following indicators: proportion of T cells, sjTREC level of naïve T cells, and DHEA-S level; the therapeutic effect can be evaluated based on one or more of the above indicators and indicators of ovarian inflammation, androgens, ovulation or metabolism.

[0087] In some implementations, the subjects are PMOS patients with at least one of the following characteristics: peripheral blood virtual memory CD8. + The proportion of T cells increased, the level of sjTREC in naive T cells decreased, and serum DHEA-S increased.

[0088] Furthermore, this application also provides a kit for screening drugs that inhibit virtual memory T cell polarization, comprising: IFN-α and IL-4 for inducing virtual memory T cell formation; and for detecting naïve CD8+. + Reagents for T cells and virtual memory T cells.

[0089] In some embodiments, the detection reagent includes: antibodies for recognizing at least three biomarkers selected from CD3, CD8, CD44, CD49d, CD45RA, NKG2A, CXCR3, and CCL5. In some embodiments, the detection reagent includes: nucleic acid detection reagents for detecting at least three biomarkers selected from CD3, CD8, CD44, CD49d, CD45RA, NKG2A, CXCR3, and CCL5.

[0090] On the other hand, this application also provides a method for screening drugs that inhibit virtual memory T cell polarization, which includes the following steps: Provides initial CD8 + The test cells containing T cells; The test cells were divided into at least a control group and a candidate group. The candidate group was contacted with the candidate, and the test cells in each group were cultured under stimulating conditions that could induce the formation of virtual memory T cells. After culture, the proportion of virtual memory T cells in CD8+ was measured in both the control group and the candidate group. + The proportion of the total number of T cells and / or the number of virtual memory T cells; Compared with the control group, the candidate group had a reduced percentage of virtual memory T cells per CD8 group. + The candidate is identified as a drug capable of inhibiting virtual memory T cell polarization when considering the proportion of the total number of T cells and / or the number of virtual memory T cells.

[0091] In this application, "candidate" refers to the material to be evaluated for its ability to regulate T. VM Any substance, preparation, or combination thereof that has cell formation, phenotype, migration, tissue homing, cytotoxicity, or inflammatory function.

[0092] In some embodiments, the stimulation conditions include IFN-α and IL-4. In some embodiments, the stimulation conditions include IFN-α and IL-4, anti-CD3 antibody, and anti-CD28 antibody.

[0093] In some implementations, the test cells are selected from mouse or human cells derived from: naïve CD8 cells. + T cells, containing naïve CD8 + T cells are peripheral blood mononuclear cells containing naïve CD8. + T cells in spleen cells containing naïve CD8 + T cells in lymph nodes, containing naïve CD8 + T cells are thymic-derived cells and T cells differentiated from pluripotent stem cells.

[0094] In some implementations, mouse virtual memory T cells utilize CD45 + CD8 + CD44 + CD49d^low phenotype recognition; human virtual memory T cells via CD3 + CD8 + CD45RA + NKG2A + Phenotypic recognition.

[0095] In some implementations, after culture is completed, one or more indicators selected from the following are also detected: CXCR3, CCL5, NKG7, PRF1, IFNG, GZMB, CD44, CD49d, NKG2A, cytokine release, cytotoxicity, migration ability, chemotaxis, myeloid cell recruitment ability, and interaction with target cells or myeloid cells.

[0096] In some implementations, candidates that can also reduce CXCR3 expression in virtual memory T cells and / or reduce the chemotactic response of the virtual memory T cells to CXCL9, CXCL10, or CXCL11 are identified as drugs that can inhibit virtual memory T cell polarization, wherein the inhibition of virtual memory T cell polarization includes inhibiting virtual memory T cell tissue homing.

[0097] In some implementations, candidates that can also reduce CCL5 expression or release from virtual memory T cells and / or reduce the ability of said virtual memory T cells to recruit myeloid cells via CCR1 and / or CCR5 are identified as drugs capable of inhibiting virtual memory T cell polarization, said inhibition of virtual memory T cell polarization including inhibition of virtual memory T cell-mediated inflammatory amplification.

[0098] In some implementations, the candidates are selected from small molecule compounds, biological products, antibodies, fusion proteins, peptides, nucleic acids, extracellular vesicles, microorganisms or microbial-derived components, nutrients, nutritionally active ingredients, natural products, plant extracts, prebiotics, probiotic metabolites, postbiotics, and combinations thereof.

[0099] In some implementations, the candidate is used to prevent, alleviate, or treat CD8-related virtual memory disorders. + Diseases characterized by abnormal T-cell formation, tissue homing, or enhanced inflammatory function, preferably PMOS or androgen excess-related immunoendocrine abnormalities.

[0100] In some implementations, the nutrients or nutritionally active ingredients are selected from amino acids and their derivatives, fatty acids, vitamins, minerals, polyphenols, flavonoids, terpenes, quinones, melatonin and its analogues, coenzymes and their precursors, nicotinamide adenine dinucleotide precursors, microbial metabolites and combinations thereof.

[0101] In some implementations, at least two concentrations of the candidate are set to obtain a dose-response relationship, and a comprehensive score is calculated based on at least two of the following: virtual memory T cell inhibition rate, cell viability, functional indicators, and consistency of repeatability experiments.

[0102] This application also provides the use of any of the aforementioned kits or methods for screening drugs that inhibit virtual memory T cell polarization in screening drugs for the prevention and / or treatment of PMOS.

[0103] Example The materials and methods used in this embodiment are as follows: Clinical specimens This was a retrospective study, using residual serum samples from women who visited the Reproductive Medicine Center of Peking University Third Hospital. Samples were derived from routine clinical testing, and no additional inclusion or exclusion criteria were set to reflect the true clinical population. All samples were delabeled before analysis. The study protocol was approved by the Institutional Review Committee of Peking University Third Hospital (Approval No.: 2019-041-02).

[0104] laboratory animals All animal experiments were approved by the Ethics Committee of Peking University Health Science Center (Approval No.: LA2022546) and conducted in accordance with institutional and national ethics guidelines. This study used C57BL / 6 mice, BALB / c mice, Foxn1 - / - Mice (BALB / c background) and Rag1 - / - Mice (C57BL / 6 background). Mice were housed in a specific pathogen-free environment with controlled temperature and humidity (29%-33%), a 12-hour light / dark cycle, and free access to food and water. Age- and sex-matched mice were used in both in vitro and in vivo experiments to avoid unintended bias. Mice involved in this study were randomly assigned to each experimental group.

[0105] DHEA-induced PMOS model To establish a DHEA-driven PMOS model, 3-week-old female C57BL / 6 mice were treated as follows: dehydroepiandrosterone (DHEA; Sigma-Aldrich, catalog number D4000) was dissolved in sesame oil (Sigma-Aldrich, catalog number S3547) and injected subcutaneously once daily for 21 consecutive days at a dose of 40 mg / kg. Each mouse received 20 μL of sesame oil per injection. Control group mice received the same volume of sesame oil as the control group.

[0106] letrozole-induced PMOS model For the aromatase inhibition model, 3-week-old female C57BL / 6 mice were administered letrozole (Sigma-Aldrich, catalog number L6545) at 1 mg / kg once daily by gavage for 21 consecutive days. Letrozole was first prepared as a stock solution in DMSO (Expandbio, catalog number BL165A) and diluted with sesame oil before use. Control group mice were given the corresponding solvent.

[0107] Vaginal smear and estrous cycle determination The estrous cycle was monitored daily via vaginal cytology examination within a specified time period. From day 11 to day 20 after the start of treatment, vaginal smears were collected daily at 9:00 AM using physiological saline. After Shor staining, the smears were examined under a microscope to determine the estrous cycle stage based on the predominant cell types. The criteria for determination were as follows: proestrus was dominated by round, nucleated epithelial cells; estrus was dominated by keratinized squamous epithelial cells; mesestrus contained epithelial cells and leukocytes; and diaestrus contained a large number of nucleated epithelial cells and numerous leukocytes.

[0108] Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) Ovaries and uterus were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 5 μm. Sections were subjected to H&E staining (hematoxylin: Origene, catalog number ZLI-9610; eosin: Origene, catalog number ZLI-9613) and immunohistochemical staining (DAB staining kit, Gene Tech, catalog number GK600705), all performed according to the manufacturer's instructions. The number of corpora lutea and cystic follicles in serial sections was quantified under blinded conditions.

[0109] T-cell clearance in the body To eliminate peripheral T cell subsets, mice were intraperitoneally injected with either the depletion monoclonal antibody CD4-In Vivo (Selleck, A2101, GK1.5) or CD8α-In Vivo (Selleck, A2102, 2.43) at a dose of 100 μg per mouse. The antibody was diluted with sterile PBS, with a final injection volume of 100 μL per mouse. The depletion antibody was administered one day before the start of DHEA treatment (day -1), followed by injections every 7 days for a total of 3 times. An isotype control antibody was used as a control.

[0110] T-cell adoptive transfer Regular memory T cells (T4) in the spleen were sorted by flow cytometry. CM ) and TVM cell populations, based on CD44 and CD49d expression. Initial CD8 + T cells were isolated from lymph nodes using a mouse initial CD8 T cell isolation kit (Biolegend, catalog number 480044) according to the manufacturer's instructions. The sorted cells were resuspended in sterile PBS and administered intravenously to each mouse on day 1 (day -1) of DHEA administration. 6 TVM cells (100 μL PBS) were administered simultaneously with DHEA treatment. TVM cells were defined as CD45 cells. + CD8 + CD44 + CD49d^low,T CM Cells are defined as CD45 + CD8+ CD44 + CD49d + .

[0111] In vitro T VM Cell induction and JAK inhibition For in vitro T VM T cells were induced and cultured with or without interferon-α (IFN-α, 50 ng / μL, ABclonal, catalog number RP01725) and interleukin-4 (IL-4, 20 ng / mL, PeproTech, catalog number 214-14-20) under stimulation with anti-CD3 (2 μg / mL) and anti-CD28 (1 μg / mL) antibodies. The JAK inhibitor baricitinib (Selleck, catalog number S2851) was prepared as a stock solution in DMSO and diluted with RPMI-1640 (Gibco, catalog number C11875500BT) immediately before use. Cells were analyzed by flow cytometry 24 hours after stimulation. In vitro experiments were independently repeated three times.

[0112] Baricitinib in vivo treatment For JAK inhibition in vivo, baricitinib (Olumiant, catalog number TA4182) was administered by gavage at a dose of 30 mg / kg once daily for 21 days, starting from the day of DHEA treatment. Baricitinib was suspended in ddH2O; the solvent control group was given ddH2O.

[0113] Flutamide treatment For androgen receptor inhibition, flutamide (Sigma-Aldrich, catalog number F9397) was first dissolved in DMSO and then diluted with sesame oil for intraperitoneal injection. Flutamide was administered at 30 mg / kg once daily, starting from the first day of DHEA treatment and continuing throughout the experimental period. Control group mice were given the corresponding solvent.

[0114] Intrathymic injection of adeno-associated virus (AAV) Three-week-old female C57BL / 6 mice were anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection). After aseptic preparation, the thymus was exposed via partial sternotomy. AAV9-shAR (shRNA sequence: 5′-AAGCACTGAAGAGACTGCTGAGTAT-3′, 2.0 × 10⁻⁶) was injected into the thymus using a Hamilton microsyringe. 12 The incision was closed with a wound clamp, and the mice recovered under standard feeding conditions. Alternatively, AAV9-EGFP (vg / mL) or the control AAV9-EGFP was injected directly into each thymic lobe (25 μL per lobe).

[0115] Rag1 - / - mouse initial CD8 +T cell regeneration and differentiation fate analysis The initial CD8 obtained from sorting + T cells adoptively transferred to Rag1 - / - Mice were DHEA-induced; the experimental group was simultaneously given baricitinib. T cells in donor cells were detected at specified time points. VM Cell ratio, T CM The proportion of cells in the phylum and transcriptional phenotype were determined to evaluate the effect of JAK repression on CD8. + The influence of T cell differentiation fate.

[0116] Human peripheral blood T VM Detection of cytomorphic cells and sjTREC Human peripheral blood T VM CD3-like cells + CD8 + CD45RA + NKG2A + A gating strategy was used for flow cytometry detection. sjTRECs in naïve T cells were quantified using nucleic acid amplification methods as a surrogate indicator of recent thymic output; the results could be analyzed in conjunction with the subject's age and DHEA-S levels.

[0117] Tissue collection and preparation of single-cell suspension Spleen and thymus: Spleen and thymus were harvested at the designated endpoint and mechanically dissociated using a 70 μm cell sieve (Biosharp, catalog number BS-70-CS) to prepare single-cell suspensions. Red blood cells were lysed using ACK lysis buffer.

[0118] Peripheral blood: Peripheral blood was collected via retroorbital sampling for flow cytometry and / or serum preparation. For flow cytometry, erythrocytes were lysed twice with ACK lysis buffer (first lysis for 30 minutes, second lysis for 10 minutes), then filtered through a 70 μm cell sieve before staining.

[0119] Lymph nodes: lymph nodes were collected and mechanically dissociated using a 70 μm cell sieve to prepare a single-cell suspension.

[0120] Ovaries: Ovaries were mixed in groups (4 or 20 per group), minced, and placed in digestion buffer containing collagenase D (0.5 mg / mL, Roche, catalog number 11088858001) and DNase I (0.1 mg / mL, Coolaber, catalog number CD4871). Digestion was performed at 37°C and 200 rpm for 30 minutes with shaking. After digestion, the cells were centrifuged and resuspended in 3 mL of FACS buffer (PBS containing 1% FBS). Lymphocytes were enriched by density gradient centrifugation using Percoll (Cytiva, catalog number 17089109): The cell suspension was added to the top layers of 4.5 mL of 80% Percoll and 4.5 mL of 40% Percoll, and centrifuged at 800×g for 20 minutes without stopping. Cells at the 40% / 80% boundary were collected as ovarian lymphocytes, centrifuged, resuspended in FACS buffer, and subjected to flow cytometry antibody staining.

[0121] Uterus: Uteruses were grouped (4 per group) and processed in a similar manner. A brief description is as follows: After mincing, the uteruses were placed in a digestion buffer containing collagenase D (1 mg / mL) and DNase I (0.5 mg / mL), and digested at 37°C and 200 rpm for 20 minutes with shaking. After digestion, the cells were filtered through a 70 μm cell sieve, centrifuged, and resuspended in FACS buffer for flow cytometry antibody staining.

[0122] Peripheral blood mononuclear cell (PBMC) isolation Human peripheral blood was collected in blood collection tubes containing EDTA. PBMCs were separated by density gradient centrifugation using Ficoll separation buffer (Dakewe, catalog number 7111011) according to the manufacturer's instructions. The separated PBMCs were washed twice with PBS and resuspended in FACS buffer for flow cytometry or single-cell RNA sequencing.

[0123] Flow cytometry For surface staining, cells were incubated with specific antibodies in FACS buffer (PBS containing 1% FBS) at room temperature for 30 minutes, followed by analysis on a BD Verse flow cytometer. Data were analyzed using FlowJo v10.8.1 software.

[0124] The antibodies used were as follows: anti-CD45 (25-0451-81, 30-F11, 1:250), anti-CD4 (35-0041-U500, GK1.5, 1:250), anti-CD8α (35-0081-U500, 53-6.7, 1:250), anti-CD44 (103036, IM7, 1:250) and anti-CD49d (103622, R1-2, 1:250) (all purchased from Biolegend).

[0125] Signal binding T cell receptor deletion loop (sjTREC) detection Cell sorting was performed on human peripheral blood (CD3) using fluorescence-activated cell sorting. + CD8 + CD45RA + NKG2A - ) or mouse peripheral blood (CD45) + CD8 + CD44 - CD49d - Separate the initial CD8 in ) + T cells. Genomic DNA was extracted using a genomic DNA extraction kit (ZOMANBIO, catalog number ZP307) according to the manufacturer's instructions. sjTRECs were quantified by real-time quantitative PCR using specific primers. Primers used for qPCR are listed in Supplementary Table 1.

[0126] RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was collected from the ovaries and uterus, extracted using TRIzol reagent (Vazyme, catalog number R401-01), and reverse transcribed using HiScriptIII All-in-one RT SuperMix Perfect for qPCR (Vazyme, catalog number R333-01) according to the manufacturer's instructions. qPCR was performed using PerfectStart Green qPCR SuperMix (+Universal Passive Reference Dye) (TransGen Biotech, catalog number AQ602) on an Applied Biosystems 7500 real-time PCR system. Relative gene expression levels were calculated using a 22 - The ΔΔCT method was used for calculation, with Rps16 as an internal control. Primers used for qPCR are listed in Supplementary Table 1.

[0127] Serum testosterone measurement Mice were anesthetized and blood was collected after a 4-hour fast. Blood was collected from the choroidal vein of the eye and centrifuged at 12,000 rpm for 20 minutes at 4°C. The separated serum was stored at -80°C for analysis. Total testosterone levels were measured using an ELISA kit (Demeditec Diagnostics, GmnH, DEV9911) according to the manufacturer's instructions.

[0128] Glucose tolerance test (GTT) Mice were fasted for 12 hours prior to the GTT. D-glucose was administered intraperitoneally at 2 g / kg. Blood was collected from the tail vein at 0, 15, 30, 60, 90, and 120 minutes, and blood glucose was measured using an Accu-Chek Performa blood glucose meter (Roche Diagnostics).

[0129] Western blot for protein immunoblotting Tissues were homogenized on ice in RIPA lysis buffer (Applygen, C1053) supplemented with a protease inhibitor mixture (Selleck, B14001). The lysis buffer was centrifuged at 12,000 × g for 15 min at 4 °C, and the protein concentration in the supernatant was quantified using a BCA protein assay kit (Thermo, 23225). Membranes were blocked with PBST containing 5% skim milk at room temperature for 1 h, followed by overnight incubation at 4 °C with anti-androgen receptor antibody (Abcam, ab133273, 1:1000) and anti-GAPDH antibody (RayBiotech, RM2002, 1:5000). Band grayscale was quantified using ImageJ software and normalized to GAPDH.

[0130] Batch RNA sequencing (RNA-seq) Total RNA was extracted from tissues using TRIzol reagent, and RNA-seq libraries were constructed using Berry Genomics. Gene expression levels were quantified using FPKM (kilobases per million fragments) and normalized according to gene length and reads. Differentially expressed gene analysis was performed using DESeq2 (v1.46.0, RRID:SCR_015687, Wald test). Principal component analysis (PCA) and Pearson correlation analysis were used to assess transcriptional similarity between samples or tissues. Heatmaps of selected genes were generated based on row-normalized z-score expression values. Functional enrichment analysis was performed using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and WikiPathways databases. Data visualization was performed using pheatmap (v1.0.12, RRID:SCR_016418) and ggplot2 (v3.5.0, RRID:SCR_014601) R packages.

[0131] Single-cell RNA sequencing (scRNA-seq) This study generated three scRNA-seq datasets, namely mouse spleen CD8. + T cells, mouse ovarian CD45 + Lymphocytes and human subject PBMCs. To obtain CD8. + T cells were obtained by mincing spleens from mice in the control and DHEA-treated groups, grinding them in pre-chilled FACS buffer, and filtering through a 70 μm cell sieve. After erythrocyte lysis, CD8 cells were enriched using a mouse CD8 T cell isolation kit (Biolegend, catalog number 480035) according to the manufacturer's instructions.+ T cells were isolated from the ovaries of mice in the DHEA-treated and control groups by flow cytometry. + Lymphocytes. PBMCs were isolated from healthy controls and PMOS patients by Ficoll density gradient centrifugation. After live cell mixing, the cells were sequenced by Berry Genomics using the 10x Genomics platform.

[0132] Raw sequencing data were processed using Cell Ranger (10x Genomics, v8.0.1, RRID: SCR_017344) to generate raw UMI count matrices. Data was converted to Seurat objects (Seurat R package v5.3.0, RRID: SCR_016341). Low-quality cells were excluded based on UMI counts, number of detected genes, mitochondrial gene content, and ribosomal gene content. Data normalization and scaling were performed using the NormalizeData and ScaleData functions, respectively. Seurat objects from the control and disease / treatment groups were merged using the merge function, and the top 3,000 hypervariable genes in the merged dataset were identified using FindVariableFeatures. Batch effects were corrected using the Harmony R package (v0.1.1, RRID: SCR_022206). Scaled data and Harmony-corrected embeddings were used for downstream analysis. Principal component analysis was used for dimensionality reduction; the number of principal components used in downstream analysis was determined based on the elbow plot. Cell clustering was performed using the FindNeighbors and FindClusters functions. The marker genes for each cluster were identified using FindAllMarkers. The clustering results were visualized using a two-dimensional UMAP plot and annotated according to the expression of typical marker genes. Cell proportions were calculated using the dplyr R package (v1.1.4, RRID:SCR_016708) and visualized using the ggplot2 R package (v3.5.0, RRID:SCR_014601).

[0133] The expression of marker genes in each annotated cell population was visualized using Seurat's DotPlot and FeaturePlot functions (v5.3.0, RRID: SCR_016341). The expression patterns of selected target genes were further visualized using the Nebulosa R package (v1.12.0). Pathway activity scores were calculated using the AUCell R package (v1.20.2, RRID: SCR_021327). Trajectory analysis of selected cell populations was performed using the monocle3 R package (v1.3.4, RRID: SCR_018685). Cell ranking along pseudo-time was based on the learned master graph and cell state annotations. Intercellular interactions were analyzed using the CellChat R package (v2.1.1, RRID: SCR_021946) based on species-matched ligand-receptor pairs in CellChatDB. The inferred intercellular communication network was visualized to compare interaction patterns among the annotated cell populations.

[0134] GO enrichment analysis GO enrichment analysis was performed using DAVID Bioinformatics ( https: / / davidbioinformatics.nih.gov / tools.jsp The assay was performed to identify pathway activities in different cell populations. Results were visualized using the ggplot2 R package (v3.5.0, RRID:SCR_014601). GO analysis was applied to the scRNA-seq data.

[0135] Statistical analysis Data are expressed as mean ± standard error. Statistical analysis was performed using GraphPad Prism (v10.5.0). Significant differences between groups were assessed using unpaired Student's t-test, one-way ANOVA, and two-way ANOVA, supplemented by Tukey's multiple comparison test and Pearson's correlation test. P < 0.05 was considered statistically significant. All experiments were independently repeated at least twice to ensure consistency.

[0136] Data availability statement The scRNA-seq and RNA-seq data from this study have been stored in the GEO database, with accession numbers GSE316046, GSE316220, GSE336822, and GSE337671. The GEO database data was downloaded from MSigDB. http: / / www.gsea- msigdb.org / gsea / downloads.jsp ).

[0137] Example Example 1: AR signaling in thymic stromal cells drives early thymic regression in PMOS To investigate the fundamental mechanisms of PMOS pathogenesis, we established a mouse model by administering dehydroepiandrosterone (DHEA; 40 mg / kg) to C57BL / 6 mice. Figure 7 a). DHEA treatment induced estrous cycle arrest, elevated circulating ovarian-derived testosterone levels, and characteristic ovarian abnormalities, including decreased corpus luteum and increased cystic follicles (a). Figure 7 bd). Pro-inflammatory cytokine genes Il1b , Il6 and Tnf Ovarian expression also increased ( Figure 7 e). These reproductive abnormalities are accompanied by significant uterine edema and inflammation, consistent with the uterine phenotype previously reported in DHEA-induced PMOS models, and are accompanied by weight gain and impaired glucose tolerance. Figure 7 fi).

[0138] To determine potential inter-organ interactions during disease progression, we compared transcriptomic changes in the ovary, uterus, thymus, brain, heart, intestine, kidney, liver, lung, and spleen after DHEA treatment. Among these tissues, the thymus and ovary showed particularly strong transcriptional homology. Figure 1 (a, b) Genes that are consistently altered include Tgfbr2 , Pbrm1 , Smad3 , Tlr3 , Edn1 , Spon2 , Blm , Btg2 and Ngfr It is mainly associated with fibrosis processes, immune regulation, and cellular stress responses. Figure 7 Consistent with this transcriptional relationship, DHEA-treated mice exhibited significant thymic atrophy, with degeneration of both the cortical and medullary compartments and a significant reduction in the total number of thymocytes. Figure 1 c). Flow cytometry further revealed dysplasia of T cell development in the thymus, characterized by CD4+. + CD8 + Loss of double-positive (DP) thymocytes, and double-negative (DN) and CD4+ cells. + or CD8 + The number of single positive (SP) cases increased accordingly. Figure 1 d). Similar thymic atrophy and altered thymic cell composition were observed in a separate letrozole-induced PMOS model. Figure 8 af).

[0139] Next, we assessed thymic function impairment in PMOS patients by measuring signal-connected T-cell receptor cleavage loops (sjTREC), a surrogate marker of recent thymic emigration. In healthy individuals, sjTREC levels in naïve T cells decline with age, supporting the effectiveness of this assay. Figure 1 e). Notably, age-matched PMOS patients exhibited significantly lower sjTREC levels than healthy controls ( Figure 1 e) indicates a decrease in thymic output.

[0140] Time-course analysis further showed that a decrease in thymus size and thymocyte number could be detected as early as day 7 after DHEA administration, followed by a decrease in sjTREC levels on day 10. Figure 8 g, h and Figure 1 f). In contrast, uterine edema appeared on day 14, while increased expression of ovarian inflammatory genes and cystic remodeling did not become apparent until day 21. Figure 8 h, i and Figure 7 d). These findings suggest that in the DHEA-induced PMOS mouse model, thymic dysfunction precedes obvious reproductive pathology.

[0141] Next, we investigated whether DHEA-induced thymic dysfunction is dependent on androgen receptor (AR) signaling. Pharmacological AR blockade with flutamide significantly prevented thymic atrophy and restored the total number of thymic cells in DHEA-treated mice. Figure 1 g). Flutamide also rescued the DP thymocyte compartments and largely restored the abnormal accumulation of DN and SP populations to normal. Figure 1 h), indicating that AR signaling mediates thymogenesis disorder. To determine the cellular compartments where AR signaling functions, we employed AAV9-mediated AR silencing. After validating the knockdown efficiency ( Figure 8 This method delivers AAV9 directly into the thymus. It significantly reduces AR expression throughout the thymus tissue but has little effect on purified thymocytes, indicating that it preferentially targets thymic stromal cells (j). Figure 8 k). In Rag1 - / - Effective AR knockdown was also observed in the thymus of mice, further supporting the non-hematopoietic target ( ). Figure 8 Furthermore, intrathymic AAV9 administration locally reduced AR expression without affecting hepatic AR levels, thus ruling out significant systemic viral dissemination. Figure 8 m).

[0142] Finally, mice underwent intrathymic AAV9-mediated AR knockdown prior to DHEA administration. Figure 1i). Control group mice showed significant thymic regression, while AR-silenced thymus was largely protected, with recovery of total thymocyte count and DP compartment, and normalization of DN and SP populations. Figure 1 (j, k). In summary, these findings suggest that DHEA activates AR signaling in thymic stromal cells, thereby disrupting thymogenesis and driving early thymic regression before the onset of obvious reproductive pathology.

[0143] Example 2: Thymic remodeling promotes T cell-mediated PMOS pathology To directly assess the contribution of the thymus to the pathogenesis of PMOS, we treated BALB / c background patients with DHEA. Foxn1 - / - (Athymic) mice, with age- and sex-matched BALB / c mice as controls. Figure 2 a). Foxn1 - / - Mice showed significantly reduced susceptibility to DHEA-induced PMOS, retained some estrus cycle, and did not exhibit significant hyperandrogenemia. Figure 2 (b, c) Histopathological analysis of ovarian tissue showed that the corpus luteum was preserved, there was no significant increase in cystic follicles, and the number of ovarian follicles increased after DHEA treatment. Il1b , Il6 and Tnf The expression remains unchanged. Figure 2 d, e). In addition, Foxn1 - / - Mice were protected from DHEA-induced weight gain and impaired glucose tolerance. Figure 2 (f, g). These findings suggest that the thymus is essential for the complete development of PMOS pathology.

[0144] because Foxn1 - / - The mice lacked both functional thymic epithelium and mature T cells, making it impossible to distinguish the contributions of thymic matrix defects and T cell defects in this model. Therefore, we used the same DHEA induction protocol as the wild-type C57BL / 6 control to investigate... Rag1 - / - Mice that retain thymic structure but lack mature T cells and B cells ( Figure 2 h). Similar to wild-type mice, DHEA treatment Rag1 - / - The mice exhibited significant thymic regression ( Figure 2 i). Unprocessed Rag1 - / - Mice exhibited well-defined, well-tissue cortical and medullary compartments, while DHEA treatment disrupted the corticomedullary structure and reduced thymic epithelial markers. Foxn1 and Aire The expression ( Figure 2 Therefore, DHEA-induced thymic remodeling still occurs even in the absence of mature lymphocytes.

[0145] Despite these thymic changes Rag1 - / - The mice remained largely resistant to DHEA-induced PMOS and maintained near-normal estrous cycles compared to wild-type mice. Figure 2 They also exhibited lower circulating testosterone levels, fewer cystic follicles, and preservation of the corpus luteum. Figure 2 m, n). In the ovary Il1b , Il6 and Tnf The expression of DHEA was significantly reduced, and DHEA-induced weight gain and impaired glucose tolerance were basically not observed. Figure 2 (oq). In short... Foxn1 - / - and Rag1 - / - The resistance in mice collectively suggests that thymic remodeling alone is insufficient to drive PMOS, and that T cells are essential to translating thymic dysfunction into reproductive, inflammatory, and metabolic pathologies.

[0146] Example 3: CD8 + T cells are the main immune effector cells in PMOS. Next, we depleted CD4 before DHEA administration. + or CD8 + T cells, to determine the T cell subsets responsible for PMOS pathology ( Figure 3 a, b). CD4 + T cell depletion failed to prevent DHEA-induced estrus cycle arrest, while CD8 + T cell depletion partially preserves the normal cyclical nature ( Figure 3 c). Similarly, CD4 + Even after T cell depletion, circulating testosterone levels remain elevated, while CD8+ levels remain elevated. + T cell depletion significantly reduces circulating testosterone levels. Figure 3 d). Histological analysis showed that CD4 + Mice with T-cell depletion retained characteristic corpus luteum loss and increased cystic follicles. Figure 3 e). In contrast, CD8 + T cell depletion preserves the corpus luteum and significantly reduces the formation of cystic follicles. Figure 3 e).

[0147] These subgroup-specific effects also extend to ovarian inflammation and metabolic dysfunction. CD8 + T cell exhaustion synergistically reduced ovarian function Il1b ,Il6 and Tnf The expression, and CD4 + T cell exhaustion only produces partial suppression, mainly affecting Il6 and Tnf ( Figure 3 f). Furthermore, DHEA-induced weight gain and impaired glucose tolerance are observed in CD4+. + T cells persist even after depletion, but can persist through CD8. + T cell exhaustion was significantly reduced. Figure 3 g, h). Consistent with these functional findings, immunostaining of ovarian tissue showed that CD8 in DHEA-treated mice was reduced. + Increased accumulation of T cells and significant infiltration around the follicular region ( Figure 3 i). In summary, these findings confirm CD8 + T cells are the main effector subset that links thymic remodeling to PMOS pathology.

[0148] Example 4: Virtual Memory CD8 + T cells preferentially proliferate during the development of PMOS. To determine the pathogenic CD8 associated with PMOS + T cell subsets, specifically CD8+ cells isolated from the spleen of C57BL / 6 mice after DHEA treatment for 21 days. + T cells underwent single-cell RNA sequencing (scRNA-seq) Figure 9 a). Based on classical transcriptional features, CD8 + T cells are divided into five subsets: virtual memory T (T1) VM ) cells, effector T cells, effector memory T cells, central memory T cells, and naive T cells ( Figure 9 b). For example Figure 4 As shown in a, DHEA treatment induces T VM Cells proliferated significantly, while the initial T cell compartments contracted.

[0149] Other CD8 + Compared to T cell subsets, T VM Cells highly express memory-related genes, such as Sell , Il7r , Cd44 and Il2rb However, low expression of the encoding CD49d It ga4 ( Figure 4 b). They also exhibit transcription factors Eomes and Tbx21 Increased expression of [certain genes], as well as increased expression of genes associated with effector function and cytokine responsiveness. Figure 4 b). Differential expression analysis further showed that T exposed to DHEAVM The cells upregulated programs associated with chemotaxis, cytotoxicity, and T cell activation, indicating that the population underwent both numerical expansion and functional activation. Figure 4 c, d). Quasi-time series analysis will use T VM The cell is located upstream of the effector T cell state. Figure 4 e), and its pathogenic effect CD8 during the development of PMOS. + Consistent with the potential contribution of T cell generation.

[0150] Next, we validated these findings using flow cytometry, specifically by introducing mouse T cells... VM Cells identified as CD8 + CD44 + CD49d low cell( Figure 9 c). Consistent with scRNA-seq results, T levels in peripheral blood, spleen, ovaries, and uterus were significantly increased after DHEA treatment. VM Cell frequency increased significantly ( Figure 4 f). It is worth noting that T VM Cellular expansion could be detected as early as day 10, coinciding with the time of decline in sjTREC levels, and preceding the appearance of obvious ovarian pathology. Figure 4 g and Figure 9 d). In the peripheral blood and spleen of letrozole-induced PMOS mice, T VM The number of cells also increased, indicating that its expansion was common in different experimental models. Figure 9 e, f).

[0151] To determine T VM Whether cells also expand in PMOS patients was determined by quantifying circulating human T cells using flow cytometry. VM These cells were defined as CD3-like cells based on previously established phenotypic criteria. + CD8 + CD45RA + NKG2A + T cells ( Figure 9 g). Healthy individuals contain only a small amount of T. VM In a subset of PMOS patients, these cells showed significant amplification in the peripheral blood. Figure 4 h). Notably, patients with elevated circulating dehydroepiandrosterone sulfate (DHEA-S) levels had elevated T... VM The cell frequency was significantly higher than that of healthy controls and PMOS patients with low DHEA-S levels. Figure 4 h). In summary, these findings determine T VM Cellular expansion is a conserved feature of PMOS and links this population to androgen excess.

[0152] Example 5: T VM Cells amplify ovarian inflammation and exacerbate PMOS pathology. To determine T VM Do cells directly exacerbate PMOS pathology? We isolated T cells from DHEA-treated mice. VM Cellular and conventional memory T (T CM Cells were adopted and transferred into naïve wild-type recipient mice, which were then stimulated with low-dose (20 mg / kg) or standard-dose (40 mg / kg) DHEA. Figure 5 a and Figure 10 a). Under low-dose DHEA treatment, control mice and T mice CM Cell receptor mice maintain normal estrous cycles, while T VM Cell receptor mice exhibited significant estrous cycle arrest ( Figure 5 (b, c). Only those who accept T VM Mice exhibited elevated circulating testosterone levels, decreased corpus luteum number, increased cystic follicle formation, and increased ovarian activity. Il1b , Il6 and Tnf Enhanced expression, weight gain, and impaired glucose tolerance ( Figure 5 dh). These findings indicate that T VM Cells exacerbate reproductive, inflammatory, and metabolic responses to suboptimal androgen exposure.

[0153] Under standard-dose DHEA treatment, all groups experienced estrous cycle arrest during androgen administration. Figure 10 b). However, after DHEA was withdrawn on day 21, the control mice and T mice... CM Cell receptor mice gradually regained their estrus cycle, while T VM Cell receptor mice failed to recover and retained PMOS-like features for at least 10 days after treatment cessation. Figure 10 c). These mice consistently exhibited elevated testosterone levels, persistent ovarian cystic remodeling, and ovarian abnormalities. Il1b , Il6 and Tnf The persistent expression of [the substance] and the unresolved metabolic abnormalities ( Figure 10 In summary, these findings indicate that T VM Cellular aggravation of PMOS-related pathology and impede disease regression after androgen withdrawal.

[0154] To investigate the immunological mechanisms behind this pathogenic effect, we studied ovarian CD45... + Immune cells were subjected to scRNA-seq to determine the immune pattern during DHEA-induced PMOS. Figure 10 i and Figure 5 As shown in i, DHEA treatment significantly increased CD8+ in the ovarian immune compartment. + The proportion of T cells. Sub-clustering analysis identified five major CD8+ cells. + T cell population, including central memory T cells, tissue-resident memory T cells, and T cells. VM Cells, ISGhigh T cells and effector T cells ( Figure 10 j). Consistent with our flow cytometry analysis, T VM Cells were selectively enriched in the ovaries of DHEA-treated mice. Figure 5 j). It is worth noting that ovarian T VM The cells exhibited marked inflammatory and cytotoxic transcriptional programs, characterized by high expression of Nkg7 , Prf1 , Ifng and Ccl5 ( Figure 10 (j) indicates that these cells acquire powerful effector functions in the ovarian microenvironment, rather than simply acting as circulating bystanders.

[0155] Next, we investigated the cellular origin of inflammatory cytokines in the ovary. Il1b and Tnf It is mainly expressed by neutrophils, while Il6 Mainly produced by monocytes and macrophages ( Figure 10 k). DHEA treatment did not substantially alter the identity of these cytokine-producing cell populations ( Figure 10 (l) indicates that ovarian inflammation is mainly caused by the activation and accumulation of myeloid cells, rather than by changes in the cell types that primarily produce cytokines.

[0156] To determine T VM We performed ligand-receptor interaction analysis to determine how cells promote this inflammatory response. For example... Figure 5 As shown in k, ovarian T VM Cells exhibited significantly enhanced interactions with monocytes / macrophages, dendritic cells, and neutrophils. Among the predicted signaling pathways, T... VM Cellular high expression Ccl5 It may promote myeloid cell recruitment through CCR1 on neutrophils and CCR5 on monocytes / macrophages and dendritic cells. Figure 5 l, m). In summary, these data indicate that ovarian-infiltrating T VM The cells established a feedforward inflammatory circuit that maintained myeloid cell accumulation and promoted persistent ovarian pathology in PMOS.

[0157] Example 6: JAK Repression Limitation T VMCellular polarization to improve PMOS-related lesions given T VM Following our investigation into the pathogenic role of cells in PMOS, we then examined the signals regulating their differentiation and whether this process could serve as a therapeutic target. In vitro, stimulation with anti-CD3 / CD28 antibody alone induced only a small number of T cells. VM Cells, and the addition of interferon-α (IFN-α) and interleukin-4 (IL-4) significantly promoted T cells, while VM Cell generation ( Figure 11 a). Since IL-4 primarily transduces signals through the JAK-STAT pathway, we investigated the effects of the JAK inhibitor baricitinib. Figure 11 As shown in b, baricitinib inhibits T in a dose-dependent manner. VM Cell differentiation.

[0158] Next, we administered baricitinib to DHEA-induced PMOS mice. Figure 6 a). This treatment significantly reduced T levels in peripheral blood and spleen. VM Cell frequency and absolute number ( Figure 6 b and Figure 11 c). This reduction is accompanied by a partial recovery of the estrous cycle, decreased circulating testosterone levels, reduced ovarian abnormalities, and decreased uterine edema. Figure 6 ce and Figure 11 d). Baricitinib also inhibited the activity of baricitinib in the ovaries. Il1b , Il6 and Tnf The expression of [the substance] reduces weight gain and improves glucose tolerance. Figure 11 (e.g.) Consistent with this, CD8 in the ovaries after treatment + T cell accumulation was significantly reduced ( Figure 6 f).

[0159] To determine whether the protective effect of JAK inhibition is achieved by altering T VM Mediated by cell differentiation, we initialized CD8 cells before DHEA administration. + T cells translocate to Rag1 - / - In mice ( Figure 6 g). Using the initial CD8 + T-cell remodeling restored susceptibility to PMOS in mice, while baricitinib significantly reduced the resulting reproductive and inflammatory abnormalities. Figure 6 (hj), supporting a T cell-dependent mechanism.

[0160] Subsequently, we examined the fate of the transferred cells on day 10. In untreated recipient mice, a significant proportion of donor CD8 cells were transferred. + T cells acquired T cells VM Phenotype ( Figure 6In contrast, JAK inhibition significantly limited T. VM Cell differentiation, and the transfer of approximately 70-80% of donor cells into T cells. CM Set up a gate area room ( Figure 6 Therefore, baricitinib not only reduces CD8 (k). + Instead of increasing the abundance of T cells, it redirects their differentiation away from T cells. VM state.

[0161] Further RNA sequencing revealed that T VM and T CM Let the phylum population represent different transcriptional CD8 groups. + T cell status ( Figure 12 ac). T VM Cells preferentially express NK-related receptors and cytotoxic effector genes, including Eomes , Klrk1 , Nkg7 , Prf1 , Ifng And granzymes, and enriched in pathways associated with T cell activation, lymphocyte-mediated immunity, and NK cell-mediated cytotoxicity. Figure 12 c, d). It is worth noting that T VM Cells highly express Cxcr3, while T cells... CM The expression of Cxcr3 was significantly reduced in the population, which was confirmed by qPCR. Figure 12 (c, e). Meanwhile, DHEA-treated mouse ovaries exhibited CXCR3 ligands. Cxcl9 , Cxcl10 and Cxcl11 Increased expression ( Figure 12 f). T VM This complementary pattern of receptor expression in cells and ligand-induced expression in the ovary suggests that the CXCR3-CXCL9 / 10 / 11 axis contributes to T VM Cells preferentially recruit and accumulate in inflamed ovarian tissue. In contrast, T cells associated with JAK inhibitors... CM Suppose that the phylum population exhibits enrichment in proliferation, metabolism, MAPK, and IFN-I responsiveness programs. Figure 12 g). Therefore, these cells are not similar to classic quiescent T cells. CM It does not represent a cell, but rather a heterogeneous non-T cell. VM The population exhibits proliferative characteristics and partial effect memory or effect characteristics. Importantly, it is related to T... VM Compared to other cells, they exhibited weaker cytotoxicity and lower CXCR3 expression.

[0162] In this application, immune dysregulation was identified as a key driver of PMOS, and key mechanistic features shared by PMOS and autoimmune-like diseases were proposed. By depicting the functional thymus-ovarian axis, this application demonstrates that thymic remodeling occurs early in disease development and precedes ovarian pathology. This temporal relationship, along with a significant reduction in disease phenotype in thymic-agnostic and adaptive immunodeficient mice, positions the thymus as an upstream regulator of PMOS-related immune activation, rather than a secondary consequence of ovarian dysfunction.

[0163] A defining characteristic of autoimmune diseases is the disruption of central or peripheral immune tolerance, typically associated with thymic dysfunction and the emergence of a population of autoreactive T cells. The findings of this application are consistent with this framework. This application demonstrates that thymic atrophy and impaired T cell development are accompanied by T cell... VM Selective expansion of cells, a subset known for enhanced cytokine responsiveness and increased autoreactive potential, was observed. These cells accumulated in germ tissues, amplifying local inflammation and maintaining disease duration, fulfilling the functional criteria for pathogenic effector cells under autoimmune-like conditions. Importantly, adoptive transfer experiments established T... VM Cells play a causal role in exacerbating PMOS pathology, and clearing CD8 + T cells or T-restriction VM Cellular polarization significantly alleviated disease manifestations. In summary, these observations support the view that PMOS involves an immune mechanism similar to that operating in autoimmune diseases, driven by thymus-dependent T cell dysregulation.

[0164] This application further elucidates the relationship between endocrine disturbances and immune activation in PMOS. Hyperandrogenemia is widely considered a core driver of PMOS pathogenesis, and androgen excess has been shown to exert broad immunomodulatory effects. This application employs two complementary androgen-driven models, including DHEA administration and aromatase inhibition, to capture key endocrine features of PMOS. In both models, thymic remodeling and T... VM Cellular expansion indicates that excessive androgen is sufficient to disrupt thymic output and remodel peripheral T cell composition. These findings suggest that androgens act not only on ovarian steroid production but also on immune development, thus linking endocrine disorders to persistent inflammatory pathology.

[0165] PMOS is a heterogeneous disease with multiple proposed pathogenic mechanisms, including obesity, insulin resistance, altered anti-Müllerian hormone signaling, and metabolic stress. While this application focuses on an androgen-driven model, these pathways are not mutually exclusive. Obesity and metabolic inflammation may further enhance immune activation, while AMH dysregulation may affect ovarian function and immune-endocrine crosstalk. Future research will need to determine whether these factors converge on the thymus-T... VMAt the cellular axis, or through parallel mechanisms. Importantly, the framework of this application provides a unified immunological context in which different endocrine and metabolic disorders may ultimately drive chronic ovarian inflammation.

[0166] The therapeutic implications discovered in this application are noteworthy. Pharmacological inhibition of JAK-STAT signaling effectively suppresses T... VM Cell production reduced CD8 + T-cell infiltration into the ovary improved endocrine, inflammatory, and metabolic abnormalities. JAK inhibitors have been used clinically in a variety of autoimmune and inflammatory diseases, suggesting that immunotargeted therapies may be repurposed for specific PMOS subtypes characterized by immune activation.

[0167] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0168] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. Use of a substance for inhibiting virtual memory T cell polarization in the preparation of a medicament for the prevention and / or treatment of polyendocrine metabolic ovarian syndrome (PMOS).

2. Use of a substance for inhibiting virtual memory T cell polarization in the prevention and / or treatment of PMOS.

3. The use according to claim 1 or 2, wherein the substance used to inhibit virtual memory T cell polarization is a JAK inhibitor.

4. The use according to claim 3, wherein the JAK inhibitor is a JAK1 and / or JAK2 inhibitor.

5. The use according to claim 4, wherein the JAK inhibitor is selected from one or more of baricitinib, ruxolitinib, momolotinib, paclinib, fenzotinib, deruxolitinib, tofacitinib, perfetinib, utpatinib, abuxitinib, frigidinib, and AZD1480, or a pharmaceutically acceptable salt thereof. Preferably, the JAK inhibitor is baricitinib or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition for the prevention and / or treatment of PMOS, comprising a substance for inhibiting virtual memory T cell polarization.

7. A method for preventing and / or treating PMOS, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of claim 6.

8. A kit for screening for a drug that inhibits virtual memory T cell polarization, comprising: IFN-α and IL-4 are used to induce the formation of virtual memory T cells; and methods for detecting naive CD8 + Reagents for detecting naive CD8 T cells and virtual memory T cells.

9. A method for screening drugs or foods that inhibit virtual memory T cell polarization, comprising the following steps: Provided are methods of enriching for naive CD8 + T cells from a test cell population. The test cells were divided into at least a control group and a candidate group. The candidate group was contacted with the candidate, and the test cells in each group were cultured under stimulating conditions that could induce the formation of virtual memory T cells. After completion of the culturing, the proportion of virtual memory T cells to the total number of CD8 + T cells and / or the number of virtual memory T cells in the control group and the candidate group is detected. when the candidate group has a decreased proportion of virtual memory T cells out of the total number of CD8 + T cells and / or a decreased number of virtual memory T cells when compared to the control group, the candidate is determined to be a drug capable of inhibiting the polarization of virtual memory T cells.

10. Use of the kit of claim 8 or the method of claim 9 for screening drugs that inhibit virtual memory T cell polarization in screening for the preparation of drugs for the prevention and / or treatment of PMOS.

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