Identification and application of novel visceral adipose tissue treg cell subsets

Abstract

The present application provides identification of a new visceral adipose tissue Treg cell subpopulation and its application. Specifically, the present application identifies two visceral adipose tissue Treg cell subpopulations: 1) ICOS hi Treg cell subpopulation that specifically highly expresses Icos, Nkg7, Itgb1, Tmsb10, Ms4a4b and Ccl5; and 2) KLRG1 hi Treg cell subpopulation that specifically highly expresses Atf3, Lmna, Klrg1, S100a6 and Cxlc2. Experimentally verified, the new Treg cell subpopulation can improve lipid metabolism, reduce body weight, reduce fat inflammation, thereby effectively improving obesity in vivo.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, and more specifically, this invention relates to the identification and application of a novel Treg cell subset in visceral adipose tissue. Background Technology

[0002] In multicellular organisms, metabolic regulation of energy management and immune regulation of resistance to external stimuli are the most fundamental requirements for maintaining life. Immune response and metabolic regulation are highly integrated and functionally interdependent. The synergistic yet antagonistic effects of immunity and metabolism work together through multiple mechanisms to maintain homeostasis. Regulatory T cells (Tregs), as a special type of immune cell, are present in almost all peripheral circulation and tissues. They mainly participate in the regulation of the body's self-tolerance and have the functions of maintaining immune homeostasis and inhibiting excessive immune activation to prevent autoimmune disorders. In the metabolic-immune regulatory loop, regulatory T cells are sensors that sense and respond to local and systemic metabolic cues, bridging energy metabolism and immune function, and maintaining metabolic homeostasis by establishing immune tolerance.

[0003] Therefore, focusing on regulatory Treg cells, the functional hub for maintaining immune homeostasis and metabolic balance, and elucidating their core mechanisms of action to establish precise intervention targets, and then developing new strategies that can bidirectionally regulate immune metabolic disorders, is a key link in the current path from basic research to new approaches for the prevention and treatment of metabolic diseases. It has significant scientific value and clinical translation prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a novel identification and application of Treg cell subsets in visceral adipose tissue.

[0005] In a first aspect of the invention, a Treg cell subset is provided, the Treg cell subset comprising: 1) ICOS hi Treg cell subsets, the ICOS hi Treg cell subsets specifically express the ICOS gene at high levels; 2) KLRG1 hi Treg cell subset, namely KLRG1 hi Treg cell subsets specifically express the Klrg1 gene at high levels; The Treg cell subset is derived from adipose tissue.

[0006] In another preferred embodiment, the ICOS hi Treg cell subsets also specifically express Ms4a4b and / or Itgb1.

[0007] In another preferred embodiment, the ICOS hi Treg cell subsets also specifically highly express: Ccl5, Tmsb10 and / or Nkg7.

[0008] In another preferred embodiment, the KLRG1 hi Treg cell subsets also specifically express high levels of Cxcl2 and / or Lmna.

[0009] In another preferred embodiment, the KLRG1 hi Treg cell subsets also specifically express Atf3 and / or S100a6 at high levels.

[0010] In another preferred embodiment, the adipose tissue is visceral adipose tissue.

[0011] In a second aspect of the invention, the use of the Treg cell subset as described in the first aspect of the invention is provided for the preparation of a drug / formulation for one or more uses selected from the group consisting of: 1) Treatment of obesity, type 2 diabetes, and / or chronic inflammation; 2) Reduce weight; 3) Lower fasting blood glucose and / or improve glucose tolerance; 4) Reduces inflammation in adipose tissue; 5) Inhibits Teff cell proliferation.

[0012] In another preferred embodiment, the treatment of obesity includes: reducing the diameter of fat cells.

[0013] In another preferred embodiment, the obesity includes hypertrophic obesity and proliferative obesity.

[0014] In another preferred embodiment, the hypertrophic obesity is characterized by an abnormally large size of adipocytes.

[0015] In another preferred embodiment, the hyperplastic obesity is characterized by an abnormally increased number of fat cells.

[0016] In another preferred embodiment, the reduction of adipose tissue inflammation includes: reducing inflammatory cell infiltration in adipose tissue and reducing the expression of pro-inflammatory factors.

[0017] In another preferred embodiment, the drug / formulation is a pharmaceutical composition comprising: 1) The Treg cell subset as described in the first aspect of the present invention; and 2) Pharmaceutically acceptable carrier.

[0018] In a third aspect of the present invention, a drug screening method is provided, the method comprising: 1) Provide candidate drugs for treating diseases selected from obesity, type 2 diabetes, and chronic inflammation; 2) Provide a Treg cell subset as described in the first aspect of the invention; and 3) Treat the Treg cell subsets with the candidate drug and detect the number of the Treg cell subsets before and after drug treatment.

[0019] In another preferred embodiment, the Treg cell subset is derived from adipose tissue.

[0020] In another preferred embodiment, the Treg cell subset is derived from visceral adipose tissue.

[0021] In another preferred embodiment, the number of the Treg cell subset is reflected by a marker that is specifically highly expressed.

[0022] In another preferred embodiment, if the number of Treg cell subsets C1 after drug treatment is greater than the number of Treg cell subsets C0 before drug treatment, and C1 / C0 is ≥1.2, preferably C1 / C0 ≥1.5, more preferably C1 / C0 ≥2, then the candidate drug indicates that it has potential therapeutic effects on the disease.

[0023] In a fourth aspect of the present invention, a prognostic method is provided, the method comprising: 1) Administer appropriate therapeutic drugs to patients suffering from diseases selected from obesity, type 2 diabetes, and chronic inflammation; and 2) Detect the number of Treg cell subsets in patients before and after drug administration, wherein the Treg cell subsets are as defined in the first aspect of the present invention.

[0024] In another preferred embodiment, the Treg cell subset is derived from the patient's adipose tissue.

[0025] In another preferred embodiment, the Treg cell subset is derived from the patient's visceral adipose tissue.

[0026] In another preferred embodiment, the number of the Treg cell subset is reflected by a marker that is specifically highly expressed.

[0027] In another preferred embodiment, if the number of Treg cell subsets C1 in a patient after drug treatment is greater than the number of Treg cell subsets C0 in a patient before drug treatment, and C1 / C0 is greater than 1.2, preferably C1 / C0 is greater than 1.5, and more preferably C1 / C0 is greater than 2, then the drug indicates that the drug has a therapeutic effect on the disease.

[0028] In a fifth aspect of the invention, a kit is provided comprising a Treg cell subset as described in the first aspect of the invention.

[0029] In another preferred embodiment, the kit comprises: 1) The Treg cell subset described in the first aspect of the present invention; and 2) The detection reagent for the Treg cell subset as described in the first aspect of the present invention.

[0030] In another preferred embodiment, the kit is used for treatment and prognosis assessment / detection.

[0031] In a sixth aspect of the invention, a method for treating a disease is provided, the method comprising administering to a subject the Treg cell subset described in the first aspect of the invention.

[0032] In another preferred embodiment, the disease is selected from obesity, type 2 diabetes, and chronic inflammation.

[0033] In another preferred embodiment, the application includes injection or infusion.

[0034] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0035] Figure 1 The presentation shows two novel Treg cell subpopulations identified by single-cell sequencing: (A) Schematic diagram of the single-cell transcriptome sequencing workflow for Treg cells in visceral adipose tissue. Two novel Treg cell subpopulations in visceral adipose tissue were identified using high-precision single-cell sequencing technology. (B) UMAP plot showing 17,702 Treg cells from different subpopulations in visceral epididymal adipose tissue. (C) Heatmap showing differentially expressed genes in clusters 0 and 1. (D) GO enrichment analysis showing differentially expressed gene enrichment pathways in clusters 0 and 1. (E) Ridge plot depicting the functional scores of Treg isotypes using AUCell markers.

[0036] Figure 2 The identification and definition of characteristic genes for two novel visceral adipose tissue Treg cell subsets were demonstrated: the characteristic genes of the two Treg cell subsets were validated, and these two Treg cell subsets were named KLRG1. hi and ICOS hiTreg cell subsets. (A) UMAP visualization showing the characteristics of single-cell sequencing cluster 0 and cluster 1 subsets. (B) Top gene expression map of cluster 0 and cluster 1 Treg subsets. (C) Violin plot showing and comparing the mRNA expression levels of Klrg1, Cxcl2, Lmna, Atf3, S100a6, Icos, Ccl5, Ms4a4b, Itgb1, and Tmsb10 with those of adipose tissue Treg cells in scRNA-seq for cluster 0 and cluster 1.

[0037] Figure 3 ICOS was displayed hi Treg cell subsets significantly improved HFHS-induced glucose and lipid metabolism disorders in mice: Eight-week-old male Rag1- / - immunodeficient mice were fed a high-fat, high-sucrose diet for 12 weeks, and different Treg cell subsets were injected via the tail vein at week 8 of intervention. (A) Different Treg cell subsets were reinfused via the tail vein into Rag1- / - immunodeficient mice, and they were fed a high-fat, high-sucrose diet for 12 weeks. Glucose tolerance and insulin sensitivity were measured, and the mice were then sacrificed for relevant analysis. (B) Body weight of mice in each group at the end of the experiment (P<0.05). (C) Epididymal adipose tissue weight of mice in each group at the end of the experiment (P<0.05). (D) Fasting blood glucose levels of mice in each group (P<0.05). (E) Glucose tolerance test (GTT) of mice in each group. (F) Area under the glucose tolerance curve of mice in each group. *Represents ICOS hi There was a significant difference between the Treg group and the non-injection group (P<0.05), # represents KLRG1. hi Treg group and ICOS hi There was a significant difference compared to the Treg group (P<0.05).

[0038] Figure 4 ICOS was displayed hi Treg cell subsets significantly ameliorated visceral fat inflammation induced by HFHS diet in mice: (A) H&E staining of epididymal fat in each group of mice. (Staining of all mouse sections was based on observations of more than 8 sections). (B) Expression levels of pro-inflammatory cytokine-related genes in visceral fat tissue of each group of mice. (C) KLRG1 hi and ICOS hi The inhibitory effect of Treg cell subsets on T cell proliferation. (D) Percentage of Teff cells proliferating after co-culturing different Treg cell subsets. * Represents comparison with ICOS hi There was a significant difference between the Treg group and the non-injection group (P<0.05), # represents KLRG1. hi Treg group and ICOShi There was a significant difference compared to the Treg group (P<0.05). Detailed Implementation

[0039] Through extensive and in-depth research, the inventors have identified a novel subset of Treg cells in visceral adipose tissue, including 1) ICOS cells that specifically and highly express Icos, Nkg7, Itgb1, Tmsb10, Ms4a4b, and Ccl5. hi Treg cell subsets; and 2) KLRG1 cells that specifically highly express Atf3, Lmna, Klrg1, S100a6, and Cxlc2. hi Treg cell subsets. Furthermore, the aforementioned Treg cell subsets were reinfused into HFHS-induced mice, and the results showed that they effectively reduced fasting blood glucose, improved glucose tolerance, alleviated fat inflammation, reduced body weight, and inhibited Teff cell proliferation in obese mice. Based on this, the present invention was completed.

[0040] the term To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0041] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0042] ICOS, or Inducible T Cell Co-stimulatory Molecule, also known as CD278 or AILIM, is a type I transmembrane glycoprotein belonging to the CD28 / B7 family. This molecule is a T lymphocyte co-stimulatory receptor expressed on the surface of activated T cells. Its ligand, ICOSL, is distributed on antigen-presenting cells such as B cells and macrophages, and its expression is typically rapidly upregulated after T cell activation. The mechanism of action of ICOS involves its interaction with its unique ligand (ICOS-L) on antigen-presenting cells (APCs). Through conserved intracellular motifs such as YMFM, IProx, and KKKY, ICOS triggers an intracellular signaling cascade, promoting T cell activation and differentiation.

[0043] Ms4a4b is a member of the MS4A family. The transmembrane 4A (MS4A) family comprises 18 members with a four-span structure in humans. These members are differentially and selectively expressed in immune-active cells, such as B cells (CD20 / MS4A1) and macrophages (MS4A4A), and can associate with and regulate the signaling activity of different classes of immune receptors, including pattern recognition receptors (PRRs) and Ig receptors. Genetic evidence from preclinical models and humans suggests that members of the MS4A family play crucial roles in various pathological settings, including cancer, infectious diseases, and neurodegenerative diseases.

[0044] Itgb1 (the integrin β1 subunit) is an important member of the integrin family, playing a crucial role in cell adhesion, signal transduction, and cytoskeleton rearrangement. At the transcriptional level, the expression of the ITGB1 gene is regulated by various transcription factors. For example, transcription factors such as NF-κB and AP-1 can promote ITGB1 transcription in response to specific cellular signaling stimuli.

[0045] Klrg1 (Kill Cell lectin-like receptor G1) is an inhibitory lectin-like type II transmembrane receptor containing a cytoplasmic immunoreceptor tyrosine inhibitory motif (ITIM). Its main component is a homodimer molecule composed of two 30 kDa-38 kDa N-glycosylated subunits. KLRG1 recognizes E-cadherin, N-cadherin, and R-cadherin; its binding inhibits NK cell effector function and T cell proliferation, and it acts as an immune checkpoint in the tumor microenvironment.

[0046] CXCL2, also known as Gro-β or MIP-2, is a member of the CXC protein chemokine family. CXCL2 is an endotoxin-induced chemokine that acts as a highly effective chemotactic inducer for neutrophils, serving as an important inflammatory mediator. CXCL2 is involved in cancer metastasis, angiogenesis, and wound healing. CXCL2 binds to the G protein-coupled receptor CXCR2 (IL-8RB) expressed on macrophages, neutrophils, and epithelial cells, and its classic function is as a chemokine, attracting neutrophils to the site of injury.

[0047] The LMNA gene is located on human chromosome 1 (1q21.2-q21.3), with a total length of 57509 bp. It encodes lamin A / C, which belongs to the V-type intermediate fibrous protein family. This gene generates multiple protein variants, including Lamin A and Lamin C, through alternative splicing, which participate in key biological processes such as maintaining nuclear membrane stability, chromatin anchoring, and DNA replication regulation.

[0048] The CCL5 gene is located on human chromosome 17, q11.2–q12, and belongs to the CC chemokine subfamily. Its protein structure contains four conserved cysteine ​​residues (the first two adjacent). This gene generates multiple transcriptional variants through alternative splicing, encoding different protein isoforms. CCL5 is a small secretory protein widely secreted by T cells, NK cells, macrophages, platelets, epithelial cells, and certain tumor cells.

[0049] TMSB10, also known as TB10, is located on chromosome 2p11.2 and primarily encodes thymosin β10, a protein widely distributed within cells that plays multiple roles. For example, as a microtubule-binding protein, it participates in the assembly and stabilization of the cytoskeleton, which is crucial for maintaining cell morphology and movement. Furthermore, thymosin β10 is involved in cell proliferation and differentiation, playing a key role, particularly in the development of the immune and nervous systems.

[0050] NKG7 (Natural Killer Cell Granule Protein 7) is located on human chromosome 19q13.41 and encodes a four-transmembrane protein that is primarily expressed in natural killer cells (NK cells) and CD8+ T cells. It belongs to the PMP-22 / EMP / MP20 protein family and functions in the plasma membrane and lysosomes of immune cells.

[0051] The ATF3 gene is located on human chromosome 1q32.3 and belongs to the basic leucine zipper (bZIP) transcription factor family. Its protein product contains an N-terminal transcription activation domain and a C-terminal bZIP domain. The bZIP domain is responsible for binding to specific DNA sequences to form homodimers or heterodimers, thereby regulating the transcription of target genes. This gene has multiple transcription variants, and different isoforms may have different physiological functions in the regulation of target genes. For example, (1) stress response: When cells are subjected to various stress stimuli, such as oxidative stress, DNA damage, inflammation, etc., the expression of ATF3 will increase rapidly to help cells adapt to these adverse conditions; (2) gene regulation: ATF3 regulates the expression of a series of genes related to cell survival, apoptosis, inflammation and metabolism by binding to specific DNA sequences; (3) cell cycle regulation: ATF3 also participates in the regulation of the cell cycle process, especially when cells face damage, it can help cells choose to repair or enter apoptosis.

[0052] S100A6, also known as 2A9, PRA, or CABP, is a member of the S100 calcium-binding protein family. Proteins in this family are known for their distinctive EF-hand domain, which allows them to bind calcium ions and perform a variety of functions within the cell. The S100A6 gene is located on human chromosome 1q21.3 and encodes a protein that plays a role in multiple cellular processes, including cell proliferation, differentiation, and apoptosis.

[0053] Treg cells in adipose tissue The response of tissue-resident Treg cells to local tissue metabolic signals is crucial for the establishment of tissue-specific immune responses and immune tolerance. Treg cells adaptively sense the physiological and pathological environment of different tissues, responding to signals from nutrients, metabolites, hormones, and growth factors in the tissue, regulating their own expansion and immune response and regulatory functions. Therefore, Treg cells in different tissues exhibit different phenotypes and functions.

[0054] Adipose tissue plays a crucial role in maintaining metabolic homeostasis by regulating bodily activities through nutrient and energy storage and endocrine regulation. Due to its rich lipid environment, adipose tissue Treg cells are uniquely adapted to the uptake of long-chain fatty acids and enhanced intracellular lipid storage. Long-chain fatty acids promote inflammation in adipose tissue. Furthermore, adipose tissue Treg cells can directly sense adipokines and pro-inflammatory cytokines produced by adipose tissue, such as leptin, adiponectin, TNF-α, and IL-6, and respond to fluctuations in these factors under the pathophysiological conditions of obesity.

[0055] In the pathological condition of obesity, high insulin and leptin levels are negatively correlated with the number of Treg cells and their ability to suppress inflammation. Conversely, glucagon increases Treg cell stability and inhibits Tconv cell proliferation, thereby improving glucose homeostasis and the inflammatory response. Furthermore, adipose tissue Treg cells can also respond to sex hormones, regulating adipose tissue inflammation and metabolic regulation in a sex-dependent manner.

[0056] In this invention, two novel subsets of Treg cells in visceral adipose tissue were unexpectedly discovered: 1) ICOS hi Treg cell subsets: specifically highly express Icos, Nkg7, Itgb1, Tmsb10, Ms4a4b, and Ccl5; and 2) KLRG1 hi Treg cell subsets: specifically highly express Atf3, Lmna, Klrg1, S100a6, and Cxlc2.

[0057] In one specific embodiment, the novel Treg cell subset was reinfused into HFHS diet-induced mice and found to improve glucose and lipid metabolism, enhance glucose tolerance, reduce body weight, decrease adipocyte diameter, reduce fat inflammation, and inhibit Teff cell proliferation.

[0058] In one specific embodiment, ICOS hi Treg cell subsets have significant effects in improving glucose and lipid metabolism, enhancing glucose tolerance, reducing body weight, decreasing adipocyte diameter, reducing fat inflammation, and inhibiting Teff cell proliferation.

[0059] Pharmaceutical Composition The present invention also provides a pharmaceutical composition. In a preferred embodiment, the pharmaceutical composition is ICOS. hi Treg cell subsets or KLRG1 hi Treg cell subsets, and pharmaceutically acceptable carriers or delivery vehicles. These substances are typically formulated in non-toxic, inert, and pharmaceutically acceptable aqueous carrier media, with a pH usually around 5-8, preferably around 6-8, although the pH may vary depending on the nature of the substance being formulated and the condition being treated.

[0060] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Injection administration preferably includes intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in liquid form, such as an aqueous solution, non-aqueous solution, or suspension, and more preferably an injection or infusion.

[0061] Pharmaceutically acceptable carriers include, but are not limited to, saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Preferably, the pharmaceutical composition / pharmaceutical combination of the present invention further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents, etc.

[0062] Preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by employing experiments not exceeding the norm. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are within the scope of a skilled physician's expertise.

[0063] Compared with the prior art, the main advantages of the present invention include: 1. This invention identifies ICOS with a unique gene expression profile in visceral adipose tissue. hi and KLRG1 hi Treg cell subsets were investigated, and their roles in regulating glucose and lipid metabolism and in obesity treatment were experimentally verified.

[0064] 2. The novel Treg cell subsets identified in this invention, whose changes in quantity and function can serve as biomarkers for assessing metabolic health and predicting the risk of obesity and type 2 diabetes, provide new avenues for cell therapy for related diseases.

[0065] 3. The relationship between the novel Treg cell subsets disclosed in this invention and obesity and obesity-related diseases can be used to assist in the in vitro screening of therapeutic drugs for these diseases, and can also serve as a potential indicator of the disease progression.

[0066] 4. Treg cell therapy has higher biocompatibility and fewer potential side effects compared to drug therapy.

[0067] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0068] Experimental materials and methods 1. Laboratory animals and food Rag1 gene knockout (Rag1) - / -The Rag1 mouse is an important model of severe combined immunodeficiency (SCID). The deletion of the Rag1 gene in this mouse leads to the loss of function of the encoded RAG1 protein (a catalytic component of the V(D)J recombinase complex), thereby preventing the mediation of V(D)J rearrangements in the T cell receptor (TCR) and B cell receptor (BCR) genes. This results in complete arrest of T and B lymphocyte development in the early stages, a lack of mature T and B cells in peripheral immune organs, and consequently, a lack of adaptive immune response in the mouse. The Rag1- / - mice used in this experiment were purchased from Shanghai Southern Model Biotechnology Co., Ltd.

[0069] The high-fat, high-sugar (HFHS) diet for the mice was purchased from Research Diet, Inc. in the United States.

[0070] 2. Main reagents, instruments and equipment Main reagents: Freestyle blood glucose meter and blood glucose test strips (Abbott); recombinant human insulin injection (EliLilly and Company); glucose (Sigma); flow cytometry antibody purchased from Thermo Scientific and BioLegend.

[0071] Instruments and equipment used: 4℃ / -20℃ freezer purchased from Haier Corporation; ultra-low temperature freezer purchased from Thermo Scientific Forma; micropipette purchased from Rainin and Eppendorf; refrigerated centrifuge purchased from Eppendorf; PCR instrument purchased from Bio-Rad; ABI 7500 Fast Real-Time PCR System purchased from Applied Biosystems; CytoFLEX S flow cytometer purchased from Beckman Coulter.

[0072] Other technical services: scRNA-seq technical services are outsourced to Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0073] 3. Single-cell transcriptome sequencing Treg cells from visceral adipose tissue of wild-type Foxp3-Cre mice fed the HFHS diet for 16 weeks were sorted using a Beckman Moflo Astrios EQ flow cytometer. Because yellow fluorescent protein (YFP) is added after the Foxp3 gene, a characteristic transcription factor of Treg cells, sorting of Treg cells was achieved through autogenous YFP fluorescence. Single-cell RNA sequencing (scRNA-seq) was performed using the 10xGenomics platform, capturing a total of 17,702 Treg cells from the visceral epididymal adipose tissue, which were then analyzed.

[0074] 4. Cell sorting (1) Remove the mouse spleen under sterile conditions, gently grind it with the plunger of a syringe, and collect the cell suspension.

[0075] (2) After lysing the cells, centrifuge and discard the supernatant, resuspend the cells, and count the cells.

[0076] (3) Prepare antibody staining mixture, add it to the blocked cells and resuspend them thoroughly, incubate at 4°C in the dark for 30 min.

[0077] (4) After washing twice, resuspend the cells, store them in the dark, and sort them by flow cytometry.

[0078] (5) After sorting, collect the ICOS and KLRG1 Treg cells and count them.

[0079] 5. Animal grouping Eight-week-old Rag1- / - male mice were intravenously injected via tail vein with flow cytometry-sorted ICOS and KLRG1 cell subsets, which were then homologously reinfused into each mouse according to their group. The reinfused cell volume was 3 x 10- 5 Each mouse was then fed a high-fat, high-sucrose diet for 12 weeks to establish a high-fat, high-sucrose diet-induced obese mouse model and disordered glucose and lipid metabolism. The grouping is shown in Table 1. Table 1. Grouping and treatment of mice Construction of experimental mouse models Mice were housed in an SPF-grade animal facility where they had free access to food and water (standard food provided by the Shanghai Laboratory Animal Center). The animal facility was maintained at a temperature of 22±3℃ and a humidity of 35±5%, with a 12-hour diurnal cycle. All experiments were conducted using 8-week-old mice.

[0080] 6. Mouse marker determination and mouse tissue collection 6.1 Fasting blood glucose and glucose tolerance test Fasting blood glucose and glucose tolerance were measured in mice after 10 weeks of experimental treatment.

[0081] Glucose tolerance test (GTT): (1) The amount of glucose used in the mouse glucose tolerance test was 1 g / kg, and a 10% glucose solution was prepared with PBS.

[0082] (2) At 17:00 the day before the experiment, go to the animal room to change the mice's cages and drinking water. They should drink water normally and fast overnight for 16 hours.

[0083] (3) Fasting blood glucose (FBG) of mice was measured at 9:00 am on the day of the experiment, and the mice were weighed.

[0084] (4) Based on the weighed mouse weight, inject the corresponding dose and volume of glucose solution (1g / kg / body weight) into the peritoneum using a 1ml syringe. Start timing after the first mouse is injected into the peritoneum, and complete the injection of one mouse every minute.

[0085] (5) Monitor and record blood glucose levels at 15, 30, 60, 90 and 120 minutes after glucose injection.

[0086] (6) The mice were fed again after the experiment was completed.

[0087] 6.2 Sacrifice of mice and collection of tissues Mouse sacrifice: Four months after the experiment, the mice were sacrificed and their tissues were collected.

[0088] Method of sacrifice: Mice were anesthetized with isoflurane to induce a deep coma. Epididymal fat was quickly harvested, photographed, weighed, fixed with formaldehyde, and stained with H&E (hematoxylin-eosin) to observe pathological changes in the adipose tissue of the experimental mice. The remaining tissue was used for RT-qPCR experiments.

[0089] 6.3 Tissue RNA extraction, reverse transcription, and RT-PCR Tissue RNA extraction: (1) Place the tissue in a 2 ml tube, add 1 ml of Trizol, and grind it in a tissue homogenizer; after washing the cells with PBS, add 1 ml of Trizol and shake thoroughly to lyse them.

[0090] (2) After lysis, add 200 µl of chloroform to 1 ml of Trizol lysis system, shake to mix thoroughly, let stand at room temperature for 10 minutes, and centrifuge at 12000 rpm for 15 minutes at 4℃.

[0091] (3) After centrifugation, aspirate the clear liquid containing RNA from the upper layer and transfer it to a new centrifuge tube. Add 500 μl of isopropanol, mix well, and let stand at room temperature for 10 minutes. Then centrifuge at 12000 rpm and 4℃ for 10 minutes.

[0092] (4) After centrifugation, discard the supernatant and leave the white precipitate. Wash the precipitate with 1 ml of 75% ethanol and place it in a centrifuge at 4℃. Centrifuge at 9000 rpm for 10 minutes.

[0093] (5) Use a 1 ml pipette to aspirate the supernatant and centrifuge at 9000 rpm for 2 minutes at 4°C. Use a 20 µl pipette to aspirate any remaining supernatant and centrifuge at 9000 rpm for 1 minute at 4°C. Use a 10 µl pipette to aspirate the remaining supernatant and dissolve the RNA in DEPC water.

[0094] (6) Measure the concentration of RNA using a nucleic acid concentration meter. Perform reverse transcription experiments using a reverse transcription kit.

[0095] RNA reverse transcription: (1) Reverse PCR using the HiScript III RT SuperMix for qPCR kit.

[0096] (2) Based on the measured concentration, the amount of RNA used was standardized: 2 µg for cell samples and 5 µg for tissue samples. The target amount of RNA sample was added and the system was brought up to 12 µl with DEPC water.

[0097] (3) Add 4 µl of 4x gDNA wiper Mix, 42°C, 2 minutes to remove residual DNA.

[0098] (4) Add 4 µl of 5x HiScript III qRT SuperMix, incubate at 37°C for 15 minutes, then at 85°C for 5 seconds to complete the inversion.

[0099] Quantitative real-time PCR (q-PCR): (1) Prepare the reaction mixture (6 µl): 5 µl of SYBR enzyme, 0.5 µl of upstream primer, 0.5 µl of downstream primer, primer concentration 10 µM. Use a 384-well plate for RT-PCR that is compatible with the machine, and add 6 µl of the reaction mixture to each well.

[0100] (2) Dilute the cDNA obtained by reverse transcription 10 times with ddH2O, add 4 µl of cDNA template to each well of the 384 plate, seal the plate and centrifuge.

[0101] (3) Place the centrifuged 384-well plate into an ABI-7900-384 real-time PCR instrument for amplification.

[0102] (4) Export the data, save the reaction program, and analyze the data.

[0103] 6.4 T cell proliferation assay: Cell Trace Violet (CTV) Cell Proliferation Coating 96-well plate: (1) Antibody coating: Anti-CD3 (2 μg / ml) and anti-CD28 (1 μg / ml) antibodies were diluted with PBS and added to 96-well plates at 100 μl / well.

[0104] (2) Incubation: Place the coated plate at 4°C overnight.

[0105] Cell sorting: (1) Cell staining: The cells were stained using antibodies: CD4-APC, CD25-PE.

[0106] (2) Cell sorting: Teff cells (CD4+CD25-YFP-) and Treg cells (CD4+CD25+YFP+) were sorted by flow cytometry.

[0107] Teff cell marker CTV: (1) Cell preparation: Teff cells were sorted, washed once with PBS, and counted. The cells were resuspended in PBS and the concentration was adjusted to 1×10⁻⁶. 6 cells / ml.

[0108] (2) Preparation of CTV working solution: Dilute 5 mM CTV stock solution in preheated PBS at a ratio of 1:1000 to obtain 5 μM CTV working solution.

[0109] (3) Staining: After centrifuging the cells, resuspend the cell pellet with an appropriate amount of CTV working solution and incubate in a 37°C water bath in the dark for 20 minutes.

[0110] (4) Stop staining: Add 4 times the volume of serum-containing 1640 medium (e.g., R10 medium) and let stand at room temperature for 5 minutes to quench the reaction.

[0111] (5) Washing and resuspending: Centrifuge to remove supernatant, resuspend cells in preheated complete culture medium, and adjust cell concentration to 2×10⁻⁶. 6 cells / ml (Teff cell suspension for co-culture).

[0112] Treg cell preparation: (1) Cell sorting and counting: Treg cells (CD4+CD25+YFP+) were obtained by sorting and cell counting was performed.

[0113] (2) Washing and resuspending: Wash cells once with PBS, centrifuge and discard the supernatant. Resuspend cells in 1640 complete medium containing primocin and adjust the concentration to 1×10⁻⁶. 6 cells / ml (Treg cell suspension for co-culture).

[0114] Co-culture and detection of Teff and Treg: (1) Prepare the culture plate: Take out the 96-well plate that has been coated overnight at 4℃ and discard the coating solution.

[0115] (2) Co-culture: 100 μl of Teff cell suspension (2×10⁻⁶) was added to the culture medium. 5 cells) and 100 μl Treg cell suspension (1×10 cells) 5 The cells were mixed and added to the wells, and the Treg:Teff ratio was set to 0:1 (control wells) and 1:2 (experimental wells).

[0116] (3) Culture and detection: After the cells were co-cultured in a 37°C, 5% CO2 incubator for about 72 hours, the cells were collected and the CTV fluorescence intensity of Teff cells was analyzed by flow cytometry to assess their proliferation.

[0117] Example 1. Single-cell sequencing identified two novel Treg cell subsets in visceral adipose tissue. Foxp3-Cre and Foxp3-Cre CREBZF were sorted by flow cytometry after 16 weeks of HFHS diet. fl / fl Treg cells from mouse epididymal adipose tissue were subjected to single-cell RNA sequencing (scRNA-seq) using the 10x Genomics platform. A total of 17,702 Treg cells from the epididymal adipose tissue were captured and analyzed. Figure 1 A). scRNA-seq identified two distinct mouse eWAT Treg cell subsets ( Figure 1BC). Pathway enrichment analysis of differentially expressed genes in the two Treg cell groups revealed that cluster 0 Treg cells were involved in signaling pathways related to intracellular metabolic processes, such as lipid metabolism regulation, ketone metabolism, oxidative stress response, and lymphocyte apoptosis. In contrast, cluster 1 Treg cells were primarily enriched in immune response pathways, including interleukin-10 (IL-10) production, positive regulation of responses to external stimuli, CD4+CD25+αβ regulatory T cell differentiation, and the JAK-STAT receptor signaling pathway. Figure 1 D). Furthermore, the ridge map depicted profile scores of lipid metabolism and adaptive immune system in two subpopulations, with cluster 0 Treg cells expressing higher levels of lipid metabolism-related genes, while cluster 1 Treg cells expressed high levels of immune regulation-related genes. Figure 1 E).

[0118] Example 2. Identification and definition of characteristic genes of two novel visceral adipose tissue Treg cell subsets Further UMAP analysis of lipid metabolism and adaptive immunity-related characteristic genes further validated the specificity of lipid metabolism and adaptive immune pathways in the two Treg cell populations. Figure 2 A). Further validation of the characteristic genes of the two Treg cell groups revealed that Treg cells in cluster 0 highly expressed Atf3, Lmna, Klrg1, S100a6, and Cxlc2, while Treg cells in cluster 1 highly expressed Icos, Nkg7, Itgb1, Tmsb10, Ms4a4b, and Ccl5. Figure 2 B), the expression levels of these characteristic genes differ in different cell subpopulations ( Figure 2 C). Based on the expression of characteristic genes and for ease of subsequent flow cytometry validation, killer cell lectin-like receptor G1 (KLRG1), a transmembrane glycoprotein associated with activation and memory phenotypes, and inducible T cell costimulator (Icos) were used as markers for cluster 0 and cluster 1, and these two Treg cell subsets were named KLRG1. hi and ICOS hi Treg cell subsets.

[0119] Example 3. ICOS hi Treg cell subsets significantly improved diet-induced glucose and lipid metabolism disorders in HFHS mice. To further investigate the function of the two Treg cell subsets, 3 x 10-10 Treg cells were injected into each eight-week-old Rag1- / - male mouse via tail vein injection. 5 The mice were infused with a subset of Treg cells and then fed a high-fat, high-sucrose diet for 12 weeks to establish a high-fat, high-sucrose diet-induced obesity and glucose and lipid metabolism disorder mouse model. Fasting blood glucose and glucose tolerance were measured in the mice after 10 weeks of HFHS diet feeding. At 12 weeks, the mice in each group were sacrificed and tissues were collected. Figure 3 A). KLRG1 hi and ICOS hi Infusion of Treg cell subsets reduced mouse body weight, and compared with the group without Treg cell infusion, ICOS hi The improvement in Treg cells was more significant (P<0.05). Figure 3 B). KLRG1 hi and ICOS hi Reinfusion of Treg cell subsets significantly reduced visceral fat weight in mice, and compared with no Treg cell infusion and KLRG1 infusion. hi Compared to the Treg cell group, ICOS hi The improvement in Treg cells was more significant (P<0.05). Figure 3 C). Fasting blood glucose and glucose tolerance (GTT) results showed that adoptive infusion of two Treg cell subsets via the tail vein could reduce fasting blood glucose levels in obese mice and effectively increase their glucose tolerance. Furthermore, ICOS hi Treg cell subsets showed better improvement than KLRG1. hi Treg cell subsets ( Figure 3 DF).

[0120] Example 4. ICOS hi Treg cell subsets significantly improved visceral fat inflammation induced by HFHS diet in mice. First, adipose tissue fixed with 4% paraformaldehyde for 48 hours was paraffin-embedded and sectioned. Then, hematoxylin and eosin (H&E) staining was used to observe the pathological changes in mouse adipose tissue. H&E staining results showed that, compared with the uninjected group, the group injected with ICOS... hi Tregs and KLRG1 hi Mice in the Tregs group showed significantly reduced adipocyte diameter and decreased inflammatory cell infiltration in adipose tissue. Simultaneously, ICOS was reinfused. hi The Treg cell group showed improved efficacy compared to the KLRG1 group in mice. hi Treg cell subsets are better ( Figure 4A). The results showed that, compared with the uninjected group, mice infused with two Treg cell subsets had significantly lower expression of pro-inflammatory cytokine-related genes and significantly higher expression of anti-inflammatory cytokine-related genes in their visceral adipose tissue. hi Treg cell subsets showed better improvement (P<0.05) Figure 4 B). In vitro inhibitory assays showed that both Treg cell subsets inhibited the proliferation of Teff cells, and were similar to KLRG1. hi Compared to Treg cell subsets, ICOS hi Treg cells exhibit stronger inhibitory function. Figure 4 CD).

[0121] discuss This study discovered two novel ICOS (Intracytoplasmic Suppurative Organisms) in visceral adipose tissue. hi Treg and KLRG1 hi Treg cell subsets, including ICOS. hi Treg cells significantly improved visceral epididymal fat hypertrophy, fasting blood glucose and glucose tolerance abnormalities induced by a high-fat, high-sugar diet in Rag1- / - mice, and significantly improved visceral fat inflammation induced by a high-fat, high-sugar diet in mice. This was achieved through ICOS... hi Co-culturing a Treg cell subset with effector T cells revealed that it significantly reduced the proliferative capacity of effector T cells, indicating a stronger inhibitory function. In summary, this novel cell subset ICOS in visceral fat... hi Treg significantly improved glucose and lipid metabolism disorders and visceral fat inflammation induced by the HFHS diet in mice.

[0122] This invention discloses a novel cell subpopulation ICOS of visceral fat. hi Treg significantly improved diet-induced glucose and lipid metabolism disorders and visceral fat inflammation. This study potentially illustrates the role of ICOS in visceral adipose tissue. hi Targeting Treg cell subsets, this study aims to regulate ICOS in visceral adipose tissue by developing small molecule drugs or using existing medications. hi The number or functional activity of Treg cells; or the use of CAR-Treg cell therapy to target visceral adipose tissue with ICOS delivery. hi Treg cell subsets are enhanced for stability and function. Both approaches are effective in preventing and treating metabolic diseases caused by overeating, such as obesity, insulin resistance, and type 2 diabetes.

[0123] The innovation of this study is mainly reflected in the following aspects: 1) For the first time, ICOS with a unique gene expression profile was identified in visceral adipose tissue using high-precision single-cell sequencing technology. hi and KLRG1 hi Treg cell subsets. Current research on cell subsets in visceral adipose tissue mainly focuses on macrophages, lymphocytes, and some immune cells. This study, however, discovered novel Treg cell subsets in visceral adipose tissue using high-precision single-cell sequencing technology, and both subsets possess unique gene expression profiles. This reveals a new dimension of heterogeneity in Treg cells residing in adipose tissue, providing new research ideas for the interdisciplinary field of immunology and metabolism.

[0124] 2) Definition of visceral fat ICOS hi and KLRG1 hi Treg subsets possess unique molecular tags that distinguish them from known Treg cells. This study discovered ICOS. hi Treg cell subsets specifically express the genes Icos, Nkg7, Itgb1, Tmsb10, Ms4a4b, and Ccl5, and KLRG1. hi Treg cell subsets specifically express the genes Atf3, Lmna, KLRG1, S100a6, and Cxlc2. This gene expression profile, a unique molecular characteristic distinguishing it from other Treg cell subsets, provides a reliable and specific molecular basis for the precise identification and isolation of these two novel Treg subsets in complex tissue microenvironments.

[0125] 3) Revealing visceral fat ICOS hi and KLRG1 hi The role of Treg cell subsets in regulating systemic glucose and lipid metabolism. This study found that obesity and metabolic disorders were successfully induced in a Rag1- / - immunodeficient mouse model via a high-fat, high-sugar diet. Subsequently, ICOS was adoptedively infused via the tail vein. hi Treg cell subsets significantly reduced visceral fat accumulation, improved fasting blood glucose levels, and effectively increased glucose tolerance in model mice. Therefore, developing a cell subset that specifically enhances ICOS is crucial. hi Small molecule drugs, antibodies, or biologics that enhance Treg cell function or promote their in vivo expansion, or ICOS targeted infusion into visceral adipose tissue using CAR-Treg cell therapy. hi Treg cell subsets can be precisely supplemented and their functional activity enhanced, which will play a certain role in the treatment of diseases such as obesity, insulin resistance, and type 2 diabetes.

[0126] 4) Clarify ICOS hi and KLRG1 hiTreg cell subsets specifically ameliorate chronic inflammation of visceral fat. This study found that in a Rag1- / - immunodeficient mouse model, a high-fat, high-sugar diet successfully induced obesity and metabolic disorder, leading to chronic inflammation of visceral fat. Subsequently, ICOS was adoptedively infused via the tail vein. hi Treg cell subsets can reduce adipocyte hypertrophy and improve inflammatory infiltration. Furthermore, ICOS injections... hi Mice with Treg cell subsets showed significantly reduced expression levels of pro-inflammatory cytokines and significantly upregulated expression levels of anti-inflammatory cytokines, indicating that ICOS hi Treg cells effectively remodel the immune microenvironment of adipose tissue, transforming it from a pro-inflammatory state to an anti-inflammatory, homeostatic state. Therefore, developing cells that can specifically enhance ICOS... hi Small molecule drugs, antibodies, or biologics that enhance Treg cell function or promote their in vivo expansion, or ICOS targeted infusion into visceral adipose tissue using CAR-Treg cell therapy. hi Treg cell subsets can be precisely supplemented and enhanced to provide a new precision intervention strategy for the treatment of metabolic diseases such as obesity, insulin resistance, and type 2 diabetes.

[0127] 5) Establish a direct translational bridge from basic research to the treatment of metabolic diseases. Link novel tissue-specific cell subpopulations discovered in basic life science research with the treatment and translation of clinical metabolic diseases. Changes in the quantity and function of identified cell subpopulations can serve as biomarkers for assessing metabolic health, predicting the risk of obesity and type 2 diabetes, and can provide new cell therapy pathways for the treatment of diseases such as obesity, insulin resistance, and type 2 diabetes.

[0128] Furthermore, its target market is broad. Firstly, the discovery and definition of novel Treg cell subsets in visceral adipose tissue will play a crucial role in research on metabolic syndrome models such as insulin resistance, type 2 diabetes, chronic inflammation, and obesity. Secondly, it allows for the development of technologies that specifically enhance ICOS. hi Small molecule drugs, antibodies, or biologics that enhance Treg cell function or promote their in vivo expansion; alternatively, cell engineering techniques can be used to construct CAR-Treg cells targeting visceral adipose tissue, which can then be expanded in vitro and reinfused with ICOS. hi Treg cells, by precisely replenishing the number of this subset in adipose tissue, thereby restoring its immunomodulatory function, improving glucose tolerance and lipid metabolism disorders, thus becoming a new drug for the prevention and adjunctive treatment of metabolic diseases caused by overeating. In clinical diagnosis, for patients with metabolic syndromes such as insulin resistance, type 2 diabetes, hyperlipidemia, obesity, and fatty liver, ICOS in visceral fat...hi The clinical level of Tregs can serve as a potential indicator for assessing disease progression, and ICOS can also be developed. hi Treg-related detection kits for rapid detection of ICOS. hi The level of Tregs, which measures their level, can be used as an alternative indicator for diagnosing the condition.

[0129] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A Treg cell subset, characterized in that, The Treg cell subset includes: 1) ICOS hi Treg cell subsets, the ICOS hi Treg cell subsets specifically express the ICOS gene at high levels; 2) KLRG1 hi Treg cell subset, namely KLRG1 hi Treg cell subsets specifically express the Klrg1 gene at high levels; The Treg cell subset is derived from adipose tissue.

2. The Treg cell subset as described in claim 1, characterized in that, The ICOS hi Treg cell subsets also specifically express Ms4a4b and / or Itgb1.

3. The Treg cell subset as described in claim 1, characterized in that, The ICOS hi Treg cell subsets also specifically highly express: Ccl5, Tmsb10 and / or Nkg7.

4. The Treg cell subset as described in claim 1, characterized in that, The KLRG1 hi Treg cell subsets also specifically express high levels of Cxcl2 and / or Lmna.

5. The Treg cell subset as described in claim 1, characterized in that, The KLRG1 hi Treg cell subsets also specifically express Atf3 and / or S100a6 at high levels.

6. The use of the Treg cell subset as described in claim 1, characterized in that, For the preparation of a drug / formulation, said drug / formulation being used for one or more purposes selected from the group consisting of: 1) Treatment of obesity, type 2 diabetes, and / or chronic inflammation; 2) Reduce weight; 3) Lower fasting blood glucose and / or improve glucose tolerance; 4) Reduces inflammation in adipose tissue; 5) Inhibits Teff cell proliferation.

7. A drug screening method, characterized in that, The method includes: 1) Provide candidate drugs for treating diseases selected from obesity, type 2 diabetes, and chronic inflammation; 2) Provide the Treg cell subset as described in claim 1; and 3) Treat the Treg cell subsets with the candidate drug and detect the number of the Treg cell subsets before and after drug treatment.

8. The drug screening method as described in claim 7, characterized in that, If the number of Treg cell subsets C1 after drug treatment is greater than the number of Treg cell subsets C0 before drug treatment, and C1 / C0 is ≥1.2, preferably C1 / C0 ≥1.5, more preferably C1 / C0 ≥2, then the candidate drug has potential therapeutic effects on the disease.

9. A method for predicting prognosis, characterized in that, The method includes: 1) Administer appropriate therapeutic drugs to patients suffering from diseases selected from obesity, type 2 diabetes, and chronic inflammation; and 2) Detect the number of Treg cell subsets in patients before and after drug administration, said Treg cell subsets as defined in claim 1.

10. A reagent kit, characterized in that, The kit includes the Treg cell subset as described in claim 1.

Images