Method for activating brain regulatory t cells for ameliorating or treating autism

Abstract

The present invention relates to a composition for treating or preventing neurodevelopmental disorders, particularly autism spectrum disorder, by selectively activating brain-resident regulatory T cells (Tregs). The present composition comprises IL-2 or an IL-2 gene (AAV-GFAP-IL-2) as an active ingredient, and induces Treg proliferation in the brain to alleviate neuroinflammation and improve behavioral defects.

Description

Method for activating brain regulatory T cells to improve or treat autism

[0001] The present invention relates to a method for activating brain regulatory T cells for the improvement or treatment of autism.

[0002]

[0003] The brain has historically been regarded as an immune-privileged organ largely protected from peripheral immune surveillance. However, new evidence highlights the existence of distinct immune niches within the central nervous system (CNS), which is populated by specialized resident immune cells. Recently, moving beyond traditional microglia, diverse immune populations—such as meningeal γδ T cells, regulatory T cells (Tregs), and B lymphocytes—have been identified as residing within CNS compartments and exerting significant influence on neurodevelopment and behavior. For example, meningeal γδ T cells regulate anxiety-like behaviors through neuronal IL-17α signaling, while meningeal Tregs have been shown to regulate olfaction and maintain brain homeostasis. Furthermore, B-1α lymphocytes directly support oligodend formation during early brain development. These findings highlight the dynamic landscape of neuroimmunological interactions involving diverse immune cell populations and significantly expand our understanding of immune influences within the CNS.

[0004] During early life, the central nervous system undergoes significant developmental processes in parallel with the immune system, creating a dynamic period in which peripheral immune cells transiently access and form developing brain tissue, particularly under inflammatory or immune-activated conditions. These early-life neuroimmunological interactions are often influenced by the functional maturation of the blood-brain barrier, are a critical factor determining long-term brain health, and are associated with neurodevelopmental disorders such as autism spectrum disorder. However, the contribution and functional role of immune cells present in the brain during these early developmental periods, and how they shape long-term brain function, are not yet well understood.

[0005]

[0006] One objective of the present invention is to provide a method for treating or preventing developmental disorders by activating brain-resident regulatory T cells through the administration of IL-2, and a pharmaceutical composition for treating or preventing immune-mediated neurological diseases such as encephalitis, Alzheimer's disease, Parkinson's disease, and traumatic brain injury, as well as developmental disorders.

[0007] Another objective of the present invention is to provide a method for treating or preventing immune-mediated neurological diseases such as encephalitis, Alzheimer's disease, Parkinson's disease, and traumatic brain injury, as well as developmental disorders, by administering an adenovirus recombinant vector containing the IL-2 gene to activate brain-resident regulatory T cells, and to provide a pharmaceutical composition for treating or preventing developmental disorders.

[0008]

[0009] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0010]

[0011] Various embodiments of the present invention are described with reference to the drawings. In the following description, for a complete understanding of the present invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to make the present invention unnecessary or obscure. Reference throughout this specification to one embodiment implies that the particular features, forms, compositions, or characteristics described in association with the embodiment are included in one or more embodiments of the present invention. Accordingly, the circumstances of the embodiments expressed at various locations throughout this specification do not necessarily represent the same embodiment of the present invention. Additionally, particular features, forms, compositions, or characteristics may be combined in any suitable way in one or more embodiments.

[0012]

[0013] Immune interactions in the developing brain shape neural circuits and lifelong functions, but the identity and developmental role of brain-resident regulatory T cells (Tregs) have not yet been fully elucidated. In this invention, we identified a previously unknown population of brain-resident regulatory T cells that is prominently formed in the early postnatal brain. These cells exhibit unique antigen-specific clonal characteristics, transcriptional specialization, and stable tissue resident status distinct from peripheral or meningeal cells. Selective depletion of neonatal brain-resident regulatory T cells disrupts central nervous system immune homeostasis, leading to persistent cortical infiltration of activated αβ T cells, microglia hyperactivation, abnormal synaptic pruning, and persistent long-term cognitive impairment that persists into adulthood. Conversely, IL-2-mediated proliferation of brain-resident regulatory T cells targeting astrocytes effectively restored central nervous system immune balance and alleviated behavioral disorders in a mouse model of autism spectrum disorder (ASD). Importantly, by applying these research findings to humans, preliminary clinical studies demonstrated that low-dose IL-2 therapy improves regulatory T cell function, reduces inflammatory responses, and significantly alleviates behavioral symptoms in patients with autism spectrum disorder. This provides the first direct clinical evidence that immune-based modulation through regulatory T cells can effectively treat neurodevelopmental disorders. This invention fundamentally advances the concept of brain immune privilege, revealing that brain-resident regulatory T cells are early neuroimmunomodulators essential for normal brain maturation, and establishes an innovative therapeutic system for immune-induced neurological diseases.

[0014] Meanwhile, Tissue-resident Tregs in peripheral tissues such as skin, muscle, and intestines act as central regulators of local immune homeostasis and tissue integrity, coordinating specialized transcriptional profiles to perform specific functions in tissue development, repair, and immune tolerance. Dysfunction or depletion of these early-life tissue-resident Tregs has been shown to result in persistent tissue dysfunction and increased susceptibility to inflammation across various organs, raising the possibility that similar mechanisms operate within the central nervous system. While recent studies have identified Tregs within central nervous system-related compartments such as the meninges, their specific locations, developmental dynamics, and neuroimmunological roles within the brain itself remain unclear. Here, the inventors demonstrate that early-life brain-resident Tregs support optimal brain development and function as critical regulators of central nervous system immune niches and neural activity, highlighting their potential as promising therapeutic targets for immune-mediated neurodevelopmental disorders.

[0015]

[0016] In the first embodiment of the present invention, a pharmaceutical composition for the treatment or prevention of developmental disorders is provided, comprising interleukin (IL) or a pharmaceutically acceptable salt thereof as an active ingredient.

[0017] In the present invention, the term “developmental disorder (neurodevelopmental disorder)” refers to a case characterized by functional delay and abnormality in attention, memory, cognition, language, problem-solving, or social interaction, where development appropriate for the age does not occur and is about 25% behind the average normal expectation in developmental tests.

[0018] The aforementioned developmental disorder is one of the most common health problems in children, exhibiting a high prevalence of approximately 5-10% of all children; however, it is not easy to detect these developmental disorders early. Unless the developmental disorder is very severe, it does not manifest well during infancy or early childhood. In particular, language disorders, hyperactivity disorders, and emotional disorders are difficult to detect before the age of 3 or 4, making early diagnosis challenging. Despite the difficulty of early diagnosis, efforts are made to identify children with these disorders early because appropriate early intervention, such as early education or rehabilitation services, can minimize the long-term disabilities that may result from them.

[0019] In addition, developmental disorders are characterized by abnormalities in the four major developmental domains: motor (gross motor, fine), language (receptive, expressive), cognitive, emotional, and social skills. They include a wide variety of disorders, such as motor developmental disorders caused by abnormal motor skill development, language developmental disorders caused by abnormal language skill development, cognitive developmental disorders caused by abnormal problem-solving skills development, and emotional and social, as well as self-help developmental disorders caused by abnormal adaptive skills development.

[0020] Furthermore, the "Developmental Disability" in this invention refers to a state in which mental and physical development has not progressed in proportion to one's age, and includes cases where a developmental screening test shows a lag of approximately 25% behind the normal expected level for that age. Generally, developmental disabilities can be classified into intellectual disability, pervasive developmental disorder, specific developmental disorder, etc., but since classification criteria vary, the above classification may differ. Common symptoms of developmental disabilities include difficulty understanding and using language, difficulty understanding overall social situations and forming interpersonal relationships, an unusual obsession with specific objects, and a pattern of repeating certain behavioral procedures. The causes of developmental disabilities are not limited to a single factor but appear in a complex manner.

[0021] Specifically, developmental disorders involve abnormalities in the four main areas of development: motor skills (gross motor, fine), language (receptive, expressive), cognitive, emotional, and social skills. While they encompass a wide variety of disorders, disorders commonly included in the category of developmental disorders generally include intellectual disability, cerebral palsy, autism spectrum disorder, developmental language disorder, functional impairment of special senses including vision or hearing, learning disabilities, and attention deficit hyperactivity disorder. Among these, cerebral palsy, intellectual disability, and functional impairment of special senses, which have a relatively low incidence but present severe disabilities, are usually detected early (average age of detection for cerebral palsy: 10 months), whereas learning disabilities and attention deficit disorder, which have a high incidence but are relatively less severe, are usually detected much later during the school-age period (average 69 months and 59 months, respectively).

[0022] For the purposes of this invention, developmental disorders may be caused by biological factors such as chromosomal abnormalities and prematurity, and environmental factors such as alcohol consumption during pregnancy, an environment of separation from parents, and parental drug addiction, but the cause is not clear.

[0023] The developmental disability in the present invention may be selected from the group consisting of intellectual disability, cerebral palsy, pervasive developmental disorder, developmental language disorder, functional impairment of special senses including sight or hearing, learning disability, attention deficit hyperactivity disorder, epilepsy, and combinations thereof, but is not limited thereto.

[0024] In addition, the aforementioned pervasive developmental disorder may be selected from a group consisting of Autism Spectrum Disorder (ASD), Asperger Syndrome, Childhood Disintegrative Disorder, Rett Syndrome, Atypical Autism Spectrum Disorder, and combinations thereof, but is not limited thereto. Furthermore, developmental disorder may include all disorders occurring in developmental domains such as perception, cognition, motor skills, and language, and may include all developmental disorders involving delays in development or function, such as learning disabilities, communication disorders, motor function disorders, cerebral palsy, genetic disorders, and chromosomal disorders (Down Syndrome, Fragile X Syndrome).

[0025] In another embodiment of the present invention, a diagnostic composition is provided in which the pervasive developmental disorder is a disorder selected from the group consisting of autism spectrum disorder (ASD), Asperger syndrome, childhood disintegrative disorder, Rett's syndrome, atypical autism spectrum disorder, and combinations thereof.

[0026] In the present invention, the “Autism Spectrum Disorder (ASD)” is one of the neurodevelopmental disorders that generally appear within the first three years of life and is characterized by deficits in social and language abilities while exhibiting repetitive interests or behaviors. According to various studies, Autism Spectrum Disorder is known to be associated with the failure of normal development of neural connections in the cerebral cortex, which consists of nerve cells called neurons. In other words, as the neural circuits of the cerebral cortex develop less than the normal growth pattern, there is a lack of important connections between cerebral cortex regions, leading to the appearance of symptoms of deficits in language, social skills, and behavior.

[0027] In the present invention, “SRS-T (Social Responsiveness Scale)” refers to a scoring method for indicating autism symptoms, which calculates symptoms unique to autism that are distinguished from other mental disorders. Specifically, it measures the content of symptom observation over two years based on responses to a questionnaire from parents or primary caregivers, and includes normalization for gender, morality, level of education, and place of residence. Thus, it is a score obtained by dividing autism symptoms into five categories, obtaining a score for each, and then summing them up.

[0028] In the present invention, “autistic mannerisms (MAN-T)” or “Autistic mannerisms” refers to a score for restricted and repetitive behaviors.

[0029] In the present invention, “Autistic Communication (COM-T)” or “Social communication” refers to a score for expressions regarding social interaction.

[0030] In the present invention, “social cognition (COG-T)”, or “social cognition”, refers to the ability to understand and process social signals and roles (cues).

[0031] In the present invention, “social awareness (AWA-T)” or “social awareness” refers to the ability to recognize signals and roles (cues).

[0032] In a second embodiment of the present invention, a pharmaceutical composition for treating or preventing developmental disorders is provided, wherein, in the first embodiment, the interleukin is one or more selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, and IL-25.

[0033] In the third embodiment of the present invention, a pharmaceutical composition for treating or preventing developmental disorders is provided, wherein in any one of the first and second embodiments, the interleukin is one or more selected from the group consisting of IL-2, IL-2 mutain, TGF-beta, and IL-10, which activate the function of regulatory T cells.

[0034] In this invention, the sustained proliferation of central nervous system resident Tregs was selectively achieved to effectively reduce neuroinflammation and improve core ASD-related behaviors. Specifically, it was demonstrated that low-dose IL-2 treatment in human ASD patients successfully enhanced the number and function of Tregs, reduced inflammatory cytokines, and significantly improved behavioral symptoms. Therefore, a person skilled in the art would understand that any cytokine capable of enhancing the number and function of central nervous system resident Tregs, particularly brain-resident regulatory T cells (Tregs), could treat or prevent developmental disorders.

[0035] In the fourth embodiment of the present invention, a pharmaceutical composition for treating or preventing developmental disorders is provided, wherein in any one of the first to third embodiments, the interleukin is low dose IL-2 (LdIL-2).

[0036] In the fifth embodiment of the present invention, in any one of the first to fourth embodiments, the low dose IL-2 is a dose of 10,000 to 30,000 IU / kg, a dose of 10,000 to 29,000 IU / kg, a dose of 10,000 to 28,000 IU / kg, a dose of 10,000 to 27,000 IU / kg, a dose of 10,000 to 26,000 IU / kg, a dose of 10,000 to 25,000 IU / kg, a dose of 10,000 to 24,000 IU / kg, a dose of 10,000 to 23,000 IU / kg, a dose of 10,000 to 22,000 IU / kg, a dose of 10,000 to 22,000 IU / kg, The dosage may be 10,000 to 21,000 IU / kg, or 10,000 to 20,000 IU / kg, but is not limited thereto.

[0037] In the sixth embodiment of the present invention, a pharmaceutical composition for the treatment or prevention of developmental disorders is provided, wherein in any one of the first to fifth embodiments, the low dose IL-2 is injected every other day for two weeks.

[0038] In the seventh embodiment of the present invention, a pharmaceutical composition for preventing or treating a developmental disorder is provided, wherein in any one of the first to sixth embodiments, the developmental disorder is a disease selected from the group consisting of intellectual disability, cerebral palsy, pervasive developmental disorder (PDD), developmental language disorder, dysfunction of special senses including vision or hearing, learning disability, attention deficit hyperactivity disorder, epilepsy, and combinations thereof.

[0039] In the eighth embodiment of the present invention, a pharmaceutical composition for preventing or treating a developmental disorder is provided, wherein in any one of the first to seventh embodiments, the general developmental disorder is a disease selected from the group consisting of autism spectrum disorder (ASD), Asperger syndrome, childhood disintegrative disorder, Rett's syndrome, atypical autism spectrum disorder, and combinations thereof.

[0040] In the ninth embodiment of the present invention, an adenovirus recombinant vector comprising an interleukin gene is provided.

[0041] In the present invention, the term “adenovirus (e.g., an adenovirus vector encoding one or more immunogens)” may be derived from any adenovirus. The adenovirus used to create the adenovirus vector provided herein may be of any suitable serotype (e.g., Ad1–Ad57). In some cases, the adenovirus may be a replication competent adenovirus. In some cases, the adenovirus may be a replication-deficient adenovirus. In some cases, the adenovirus may be capable of infecting human cells (e.g., a human adenovirus). In some cases, the adenovirus may be capable of infecting non-human cells, such as chimpanzee cells (e.g., a non-human adenovirus). Examples of adenoviruses that can be used to produce the adenovirus provided herein include, but are not limited to, Ad5 adenovirus, Ad6 adenovirus, ChAdOx1, and ChAdOx2.

[0042] In this invention, the type of vector is not limited to DNA vectors, RNA vectors, or viral vectors as long as it can deliver IL-2 to the brain. This is because IL-2 can treat or prevent developmental disorders, including autism spectrum disorder, by crossing the blood-brain barrier (BBB) ​​and activating Tregs within the brain.

[0043] In the present invention, the "vector" is a nucleic acid molecule capable of transporting another nucleic acid to which a certain nucleic acid molecule is connected. One type of vector is a "plasmid," which refers to circular double-stranded DNA to which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector to which additional DNA segments can be ligated to the viral genome. Some vectors can replicate autonomously in the host cell to which they are introduced (e.g., bacterial vectors are episomal mammalian vectors with a bacterial replication origin). Other vectors (e.g., non-episosomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell and thereby replicate along with the host genome. Furthermore, some vectors can direct the expression of genes to which they are connected at the operational level. Such vectors are referred herein as "recombinant expression vectors" or simply "expression vectors." Generally, expression vectors useful in recombinant DNA techniques often exist in the form of plasmids. In this specification, "plasmid" and "vector" may be used interchangeably because plasmid is the most commonly used form of vector.

[0044] Specific examples of the expression vector in the present invention may be selected from the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, and Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμμ, T-even, T2, T3, and T7; and plant viruses, but are not limited thereto. Any expression vector known to those skilled in the art as an expression vector may be used in the present invention, and the selection of the expression vector depends on the properties of the target host cell. When introducing the vector into the host cell, it may be performed by calcium phosphate transfection, viral infection, DEAE-dextran regulated transfection, lipofectamine transfection, or electroporation, but is not limited thereto. Those skilled in the art may select and use an introduction method suitable for the expression vector and the host cell to be used. Preferably, the vector contains one or more screening markers, but is not limited thereto. Screening is possible based on whether a product is produced using a vector that does not contain screening markers. The selection of screening markers is performed by the target host cells, and since this utilizes methods already known to those skilled in the art, the present invention is not limited thereto.

[0045] To facilitate the purification of the nucleic acid molecule of the present invention, a tag sequence may be inserted into an expression vector and fused. The tag includes, but is not limited to, a hexahistidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag known to those skilled in the art that facilitates purification may be used in the present invention.

[0046] In the 10th embodiment of the present invention, an adenovirus recombinant vector is provided, wherein, in the 9th embodiment, the interleukin is one or more selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, and IL-25.

[0047] Cytokines are cell signaling molecules involved in the regulation of the immune system. For example, IL-2 is an essential cytokine involved in the proliferation and activation of T cells. IL-2 stimulates the proliferation and differentiation of T cells, induces the production of cytotoxic T lymphocytes (CTLs), differentiates peripheral lymphocytes into cytotoxic cells and lymphokine-activated killer cells (LAKs), and stimulates the proliferation and activation of NK cells (natural killer cells).

[0048] Meanwhile, there are three types of IL-2 receptors: high-affinity, intermediate-affinity, and low-affinity. High-affinity receptors consist of three subunits: IL-2 receptor alpha (IL-2Rα; CD25), beta (IL-2Rβ; CD122), and gamma (IL-2Rγ; CD132). Intermediate-affinity receptors consist of IL-2Rβ and IL-2Rγ, while low-affinity receptors consist solely of IL-2Rα. Intermediate-affinity receptors, composed of β and γ subunits, have an affinity for IL-2 approximately 100 times lower than high-affinity receptors composed of α, β, and γ subunits, but they can transmit signals upon binding to IL-2. The α-subunit confers high-affinity binding ability to the receptor but is not essential for signal transmission.

[0049] Furthermore, IL-2 mediates activation-induced cell death (AICD) in T cells. AICD is a process in which fully activated T cells undergo programmed cell death, leading to immune tolerance not only to normal self-antigens but also to persistently present antigens such as tumor antigens. IL-2 is also involved in the maintenance of peripheral CD4+CD25+ regulatory T cells (Tregs). IL-2Rα is permanently expressed on regulatory T cells. Regulatory T cells inhibit the function of cytotoxic T cells in attacking self-antigens or tumor cells. Due to these multifaceted actions of IL-2, it is not suitable for exhibiting optimal tumor-suppressing effects.

[0050] In the present invention, “interleukin-2,” “IL-2,” “IL-2 protein,” or “IL-2 polypeptide” comprises IL-2 derived from vertebrates, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise noted. This includes unprocessed IL-2 as well as any form of IL-2 processed in cells. The term also includes naturally occurring variants or “IL-2 mutants” as defined below. Full-length human IL-2 refers to mature human IL-2 of natural length, which is the molecule having 133 amino acids of SEQ ID NO. 1. Unprocessed human IL-2 further comprises 20 N-terminal amino acids not present in the mature IL-2 molecule.

[0051] In addition, the aforementioned “IL-2 (interleukin-2)” is a proteinaceous cytokine with a relatively molecular weight of about 15.5 kDa, which primarily plays a role in regulating the proliferation and activation of immune cells (especially T cells). However, because the blood-brain barrier (BBB) ​​selectively allows substances to pass through hydrophobic small molecules (generally 400–500 Da or less) or special carriers, macromolecular proteins such as IL-2 can hardly pass through the BBB.

[0052] Specifically, the BBB consists of brain capillary endothelial cells, basement membranes, and astrocyte end-foots connected by tight junctions, which strongly restricts the diffusion of hydrophilic or macromolecular substances. IL-2 cannot cross these structural barriers by passive diffusion, and there are no specific receptor-mediated transporters or transcytosis pathways.

[0053] In the 11th embodiment of the present invention, an adenovirus recombinant vector is provided, wherein in any one of the 8th to 10th embodiments, the interleukin is one or more selected from the group consisting of IL-2, IL-2 mutain, TGF-beta, and IL-10, which activate the function of regulatory T cells.

[0054] In the present invention, the sustained proliferation of central nervous system resident Tregs was selectively achieved to effectively reduce neuroinflammation and improve core ASD-related behaviors. Specifically, it was demonstrated that low-dose IL-2 treatment in human ASD patients successfully enhanced the number and function of Tregs, reduced inflammatory cytokines, and significantly improved behavioral symptoms. Therefore, a person skilled in the art would understand that a recombinant vector containing cytokine genes capable of enhancing the number and function of central nervous system resident Tregs, particularly brain-resident regulatory T cells (Tregs), could treat or prevent developmental disorders.

[0055] In the 12th embodiment of the present invention, an adenovirus cell line transfected with an adenovirus recombinant vector of any one of the 9th to 11th embodiments is provided.

[0056] In the present invention, the "host cell" includes an individual cell or cell culture that may or was a recipient of a vector(s) for the incorporation of a polypeptide insert. The host cell includes progeny of a single host cell, and said progeny may not necessarily be completely identical to the original parent cell (morphologically or in genomic DNA complements) due to natural, accidental, or intentional mutations. The host cell includes a cell transfected in vivo with the polypeptide(s) of the present invention.

[0057] In the present invention, the host cell may include cells of mammalian, plant, insect, fungal, or cellular origin, such as, for example, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; fungal cells such as yeast cells and Pichia pasteoris; insect cells such as Drozophylla and Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mice myeloma), human lymphoblastoid, COS, NSO (mice myeloma), 293T, Bow melanoma cells, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells) or PERC.6 (human retinal cells); or plant cells, but is not limited thereto, and any cell that can be used as a host cell line known to those skilled in the art may be used.

[0058] In the 13th embodiment of the present invention, in the 12th embodiment, the adenovirus cell line is provided as an adeno-associated virus (AAV).

[0059] In the 14th embodiment of the present invention, a pharmaceutical composition for preventing or treating developmental disorders is provided, comprising an adenovirus recombinant vector of any one of the 9th to 11th embodiments as an active ingredient.

[0060] In another embodiment of the present invention, a pharmaceutical composition for preventing or treating a developmental disorder is provided, wherein the developmental disorder is a disease selected from the group consisting of intellectual disability, cerebral palsy, pervasive developmental disorder (PDD), developmental language disorder, dysfunction of special senses including sight or hearing, learning disability, attention deficit hyperactivity disorder, epilepsy, and combinations thereof.

[0061] In another embodiment of the present invention, a pharmaceutical composition for preventing or treating a developmental disorder is provided, wherein the general developmental disorder is a disorder selected from the group consisting of autism spectrum disorder (ASD), Asperger syndrome, childhood disintegrative disorder, Rett's syndrome, atypical autism spectrum disorder, and combinations thereof.

[0062] Furthermore, Autism Spectrum Disorder (ASD) is a congenital developmental disorder characterized by a lack of verbal and nonverbal social skills, repetitive behaviors, and hypersensitivity to specific stimuli. Except for some patients with genetic factors, it is triggered by environmental factors during pregnancy; although diagnosis typically occurs between the ages of 2 and 3 and causes lifelong suffering, diagnostic markers remain unclear beyond current questionnaire-based diagnosis. While SSRIs (selective serotonin reuptake inhibitors) and SGAs (second-generation antipsychotics) exist as treatments, research into new diagnostic methods and treatments is necessary due to various side effects and limited efficacy.

[0063] It is receiving attention that, in addition to outwardly visible behavioral characteristics, patients with ASD suffer from unexplained digestive disorders, abdominal pain, and immune-related diseases. In fact, the global distribution of ASD patients is similar to that of people with autoimmune diseases, and conversely, mental illnesses have significantly increased in infectious diseases such as COVID-19. This suggests that there is an influence on neurobehavioral mechanisms caused by immune hyperactivity.

[0064] In the 15th embodiment of the present invention, a pharmaceutical composition for preventing or treating a developmental disorder is provided, wherein in any one of the 9th to 14th embodiments, the developmental disorder is a disease selected from the group consisting of intellectual disability, cerebral palsy, pervasive developmental disorder (PDD), developmental language disorder, dysfunction of special senses including sight or hearing, learning disability, attention deficit hyperactivity disorder, epilepsy, and combinations thereof.

[0065] In the 16th embodiment of the present invention, a pharmaceutical composition for preventing or treating a developmental disorder is provided, wherein in any one of the 9th to 15th embodiments, the general developmental disorder is a disease selected from the group consisting of autism spectrum disorder (ASD), Asperger syndrome, childhood disintegrative disorder, Rett's syndrome, atypical autism spectrum disorder, and combinations thereof.

[0066]

[0067] In addition, the composition provided in the present invention may be used as a pharmaceutical composition or a food composition, but is not limited thereto.

[0068] The "prevention" of the present invention may include, without limitation, any act that can block, suppress, or delay symptoms caused by autism spectrum disorder using the composition of the present invention.

[0069] The "treatment" and "improvement" of the present invention may include, without limitation, any act that enables symptoms caused by autism spectrum disorder to be improved or benefited by using the composition of the present invention.

[0070] In the present invention, the pharmaceutical composition may be characterized in that it is in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized in that it is intended for humans.

[0071] The pharmaceutical composition of the present invention is not limited to these, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. For injectable preparations, it may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., in combination; and for topical administration, it may include bases, excipients, lubricants, preservatives, etc. The formulations of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it can be manufactured in the form of unit dosing ampoules or multiple dosing ampoules. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release formulation, etc.

[0072] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0073] The routes of administration of the pharmaceutical composition according to the present invention are not limited to but include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0074] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0075] The pharmaceutical composition of the present invention may vary depending on several factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, elimination rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, and may be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be administered once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0076] A food composition containing the composition of the present invention as an active ingredient can be manufactured in the form of various food products, such as beverages, chewing gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, etc. Since the food composition of the present invention is composed of plant extracts that have almost no toxicity or side effects, it can be used safely even when taken for a long period for preventive purposes.

[0077] When the composition of the present invention is included in a food composition, the amount may be added in a ratio of 0.1 to 50% of the total weight.

[0078] Here, when the above food composition is prepared in the form of a beverage, there are no special limitations other than containing the above food composition in the indicated proportions, and it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in ordinary beverages. That is, as natural carbohydrates, it may include monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, conventional sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. Examples of the above flavoring agents include natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.).

[0079] In addition, the food composition of the present invention may contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0080] These components may be used independently or in combination. The proportion of these additives is not particularly important, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

[0081] The present invention has significantly advanced treatment strategies for autism spectrum disorder (ASD) by being the first to demonstrate therapeutic efficacy targeting regulatory Treg cells in both preclinical and clinical settings. Specifically, by utilizing astrocyte-targeted IL-2 gene delivery (AAV-GFAP-mIL-2), the invention selectively achieved sustained proliferation of central nervous system resident Tregs in a BTBR mouse model, effectively reducing neuroinflammation and improving core ASD-related behaviors. This suggests the potential for clinical low-dose IL-2 treatment to similarly proliferate brain-resident Tregs in humans. In other words, it demonstrated that low-dose IL-2 treatment in human ASD patients successfully improved the number and function of Tregs, reduced inflammatory cytokines, and significantly improved behavioral symptoms. Unlike conventional systemic IL-2 therapy, the present invention is characterized by directly regulating central nervous system immune homeostasis by inducing the selective proliferation of brain-resident Tregs through GFAP promoter-based astrocyte-targeted IL-2 expression.

[0082]

[0083] Figure 1 shows single-cell mappings of the brain immune environment according to developmental stages.

[0084] Figure 1a is a representative image of CD45⁺ immune cells (arrowheads) observed in the extravascular brain parenchyma of E16, P8, and P24 mice.

[0085] Figure 1b shows immune cells isolated from the brain and liver for scRNA-seq analysis.

[0086] Figure 1c shows UMAP derivatives generated from scRNA-seq of immune cells. Density plots of cells isolated from the brain and liver / spleen are shown below. NK, Natural Killer cells; ILC, Innate Lymphocytes; DC, Dendritic cells.

[0087] Figure 1d is a dot plot showing the expression of standard marker genes across immune cell populations.

[0088] Figure 1e is a UMAP density plot showing the distribution of immune cells according to the developmental stage.

[0089] Figure 1f shows the relative proportion of immune cells in the brain at each developmental stage.

[0090] Figure 1g shows the flow cytometry analysis of B cells, γδ T cells, αβ T cells, and CD4⁺ T cells according to developmental stage. Number of mice per time point n = 4–7.

[0091] The left diagram of Fig. 1h is a UMAP showing the subclustering of CD4⁺ T cells derived from the brain and spleen, and the right diagram is a density plot of brain and liver / spleen-derived cells showing Treg marker gene (Foxp3, Ikzf2) expression.

[0092] Figure 1i is a representative immunofluorescence image showing Tregs in the choroid plexus and parenchyma of the brain.

[0093] Figures 1j and 1k represent the proportion (j) and absolute number (k) of Tregs in the brain at each developmental stage. Number of mice per time point, n = 4–7. ** P < 0.01 relative to all other time points, one-way ANOVA with Tukey's multiple comparison test.

[0094] Figure 11 shows the results of a representative flow cytometry analysis of Treg cell populations extracted from various organs of P8 mice. The figure in panel b was created using BioRender.

[0095] Figure 2 shows diagrams illustrating how brain Tregs establish the identity of tissue-resident T cells early in life.

[0096] Figure a shows the results of isolating TCRβ⁺ T cells from the brains and spleens of 1W and 12W mice for scRNA-seq and scTCR-seq.

[0097] Figure 2b shows the results of UMAP analysis after Treg cell reclustering. Five subgroups were identified: dormant, active, Th1-like, proliferative, and recirculation phases. Density plots for each condition are shown on the right.

[0098] Figure 2c is a DEG heatmap of the entire Treg cluster.

[0099] Figure 2d is a diagram showing the expression levels of representative genes (Bcl2, Rora, Lgals3, Mki67, Klrg1) in brain and spleen Tregs.

[0100] Figure 2e is a density plot of gene signature scores corresponding to the dormant phase, interferon reactant phase, recovery phase, Th1-like phase, and tissue-resident Treg.

[0101] Figure 2f shows the PCA location of brain Tregs compared to other tissue-resident Treg populations.

[0102] Figure 2g is a Volcano plot showing DEGs between 1W and 12W brain Tregs within the "activated" and "TH1-like" clusters.

[0103] Figure 2h shows the results of a gene ontology abundance analysis highlighting biological pathways abundant in 1W versus 12W brain Tregs (provided by Metascape).

[0104] Figure 2i is a dotted line plot showing the expression of representative genes in 1W and 12W brain Tregs.

[0105] Figure 2j shows the gene signature scores for proliferation, OXPHOS, and tissue residence. ****P < 0.0001, Kruskal-Wallis test using Dunn's multiple comparison test.

[0106] Figure 2k shows the flow cytometry results showing higher expression of Ki-67, ICOS, and CD69 in brain Tregs compared to spleen Tregs. n = 4–5 for each group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, one-way ANOVA with Tukey's multiple comparison test.

[0107] Figure 21 is a UMAP plot showing cloned Tregs within the brain.

[0108] Figure 2m is a bar graph quantifying Treg clonality between identified clusters. The graph is expressed as mean ± standard error (SEM). The diagram in panel a was generated in BioRender.

[0109] Figure 3 shows the results indicating that a decrease in neonatal Treg induces T cell infiltration into the brain.

[0110] Figure 3a shows the experimental process of removing Treg by injecting PBS (n = 6) or DT (n = 4) into P8 and P15 of Foxp3-DTR mice.

[0111] Figure 3b shows the absolute number of lymphocytes in the dura mater and brain at 4 weeks of age.

[0112] Figure 3c shows the number and percentage of immune cell populations in the brain after Treg reduction.

[0113] Figure 3d shows that immunohistochemical staining results indicate that the majority of CD45⁺ immune cells in the brain parenchyma are CD3⁺ T cells. The graph shows the percentage of T cells among CD45⁺ immune cells (n = 3). ** P < 0.01, one-way ANOVA with Tukey multiple comparison test.

[0114] Figure 3e is a representative image of CD45⁺ immune cell infiltration in various cortical regions. P, posterior; A, anterior

[0115] Figure 3f is a spatial density map of immune cells superimposed on the Allen Brain Atlas; the posterior ampulla (RSP) is indicated by red dots. Data represent the mean of n = 3 mice.

[0116] Figure 3g shows the results of quantifying the distribution of immune cells across the cortical region (n = 3). The top-left image shows a representative 3D reconstruction of infiltrated immune cells. ** P < 0.01 compared to all other regions excluding somatosensory and visual regions; density P < 0.05 compared to all other regions, one-way ANOVA with Dunnett's multiple comparison test.

[0117] Figure 3h shows the experimental process of isolating spleen and brain infiltrating T cells and performing ex vivo stimulation after early Treg depletion.

[0118] Figure 3i shows the results of multi-cytokine analysis of culture supernatants after anti-CD3 / CD28 stimulation of brain infiltrators and splenic T cells. n = 3, collected from 1–2 mice, respectively.

[0119] Figure 3j shows the quantification and percentage of cytokine-producing CD4⁺ and CD8⁺ T cells after 4 hours of PMA / ionomycin stimulation. Not significant in b, c, j, and ns; * P < 0.05, ** P < 0.01; *** P < 0.001, two-sided Student's t-test. Graphs are presented as mean ± standard error (SEM). Dots represent individual mice.

[0120] Figure 4 shows results indicating that brain Treg loss causes microglia activation and abnormal synaptic pruning, leading to spatial memory impairment.

[0121] Figure 4a is a representative immunofluorescence image of IBA1⁺ microglia in PBS and DT-injected mice.

[0122] Figure 4b shows the results of Sholl analysis showing changes in microglia morphology after Treg depletion.

[0123] Figure 4c shows the results of quantifying microglia branching complexity at a distance of 26 μm from somatic cells. Number of cells n = 13 for 3 mice per group.

[0124] Figure 4d shows representative flow cytometry plots of microglia and the results of MHC-I and MHC-II level quantification. 4 individuals per group.

[0125] Figure 4e shows representative images and quantification results of PSD-95⁺ synaptic sites within IBA1⁺ microglia. Cell count n = 8 for 2 mice per group upon PBS administration, and cell count n = 11 for DT administration.

[0126] Figure 4f shows representative images and quantification results comparing PSD-95⁺ Punta in immune cell-infiltrated areas and adjacent non-infiltrated cortical areas (n = 5). * P < 0.05, two-sided paired t-test.

[0127] Figure 4g shows the experimental process of culturing primary microglia with brain-infiltrating T cells from DT-injected mice or spleen T cells isolated from PBS-injected embryos.

[0128] The left plot of Fig. 4h is a histogram showing the uptake of pHrodo-labeled synaptosomes by microglia, and the right plot is the result of quantifying synaptosome phagocytosis and MHC-I / II expression under co-culture conditions. T cells collected from 1-2 mice were used, with n = 6 per group. ** P < 0.01; *** P < 0.001; **** P < 0.0001, one-way ANOVA using Tukey's multiple comparison test.

[0129] Figure 4i shows the experimental process of performing Neuropixels recording and behavioral testing after early Treg depletion.

[0130] Figure 4j shows the neural firing rates and representative spike histograms by group. 4 mice per group, n = 219 cells for PBS and n = 212 cells for DT. ** P < 0.01; Kolmogorov-Smirnov test.

[0131] The left diagram in Fig. 4k shows the results of a new object / location recognition test evaluating memory. n = 19 for PBS and n = 20 for DT. The right diagram shows the results of a Y-maze test displaying the rate of spontaneous switching and the frequency of direct re-entry. n = 7 for PBS and n = 9 for DT.

[0132] Figure 5 shows the results demonstrating that brain Treg cell enhancement reshapes the immune microenvironment and alleviates ASD-like behavior in BTBR mice.

[0133] Figure 5a shows the experimental process of injecting AAV-PHP.eB-GFAP-mIL-2 or a control (EGFP) vector into BTBR mice and performing behavioral tests in adults.

[0134] Figure 5b shows the results illustrating the increase in the number of brain Treg cells after AAV-GFAP-mIL-2 treatment. n = 9 per group.

[0135] Figure 5c shows the results of an open experiment demonstrating a reduction in anxiety-like behavior after brain Treg cell enhancement. EGFP n = 17, mIL-2 n = 19.

[0136] Figure 5d shows the results demonstrating a reduction in compulsive behavior after brain Treg cell enhancement. EGFP n = 17, mIL-2 n = 18.

[0137] Figure 5e shows the results illustrating increased social interactions after brain Treg cell enhancement. EGFP n = 19, mIL-2 n = 19.

[0138] Figure 5f shows the results illustrating the correlation between brain Treg percentage and total repetitive behavior time. The values ​​were determined using the Spearman correlation coefficient.

[0139] Figure 5g shows the experimental process of isolating PBMCs from ASD patients and typical developmental control groups (TDC) for flow cytometry analysis.

[0140] Figure 5h shows the ratio of total Treg and inactive Treg to the TDC (n = 43) versus ASD (n = 76) groups.

[0141] Figure 5i shows the results for IFN-γ⁺ and GZMB⁺ CD8⁺ T cells in the TDC (n = 43) and ASD (n = 76) groups.

[0142] Figure 5j shows the results of the correlation analysis illustrating the association between immune parameters and ASD severity scores. * P < 0.05, ** P < 0.01, *** P < 0.001, Spearman correlation test.

[0143] Figure 5k shows the results of low-dose IL-2 therapy in patients with ASD (n = 24).

[0144] Figure 51 shows the results of longitudinal flow cytometry analysis of PBMCs showing an increase in peripheral Treg after IL-2 treatment.

[0145] Figure 5m shows the results of improvement in ASD behavior scores after treatment. For l and m, * P < 0.05, ** P < 0.01, analysis of mixed effects using Tukey's multiple comparison test.

[0146] Figure 5n shows the results regarding the correlation between changes in Treg frequency and changes in ATEC scores. The values ​​were determined using the Spearman correlation coefficient. The graph is displayed as mean ± standard deviation (SEM).

[0147]

[0148] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0149]

[0150] [Example]

[0151] [Experimental Method]

[0152] 1. Mouse

[0153] All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University and were performed in accordance with institutional guidelines (Yonsei Institute of Biomedical Research, Department of Animal Science). Wild-type C57BL / 6J and Slc-ICR mice were purchased from Central Research Institute Animal Corp. (Seoul, Korea), and the following strains were purchased from Jackson Laboratory: JAX#016958, JAX#016961, JAX#006148, and JAX#00282. Mice were housed under specific pathogen-free (SPF) conditions in individual ventilated cages at 23 ± 2 ℃ with a 12-hour light-dark cycle and 60 ± 10% relative humidity. Food and water were provided at random. Unless otherwise noted, both male and female mice were used in all experiments. The experimental groups were matched for age and sex.

[0154]

[0155] 2. Mouse treatments

[0156] (1) Treg depletion

[0157] Foxp3 to deplete Tregs during the neonatal period DTR25 ng of DT (Sigma-Aldrich, D0564) per 1 g of body weight was dissolved in phosphate-buffered saline (PBS) at 2.5 ng / μL and injected intraperitoneally into mice. Only PBS was administered to the control group. Injections were given at 8 months and 15 months of age.

[0158] For DT administration in cisterna magna, 8-month-old Foxp3 DTR The pups were anesthetized with isoflurane (3% for induction, 1–2% for maintenance). After a central scalp incision, 2 μL of DT (2.5 ng / μL in PBS) was injected into the logarithmic bath at a rate of 1 μL / min using a LEGATO® 130 syringe pump (KD Scientific) and a 33-gauge needle. To prevent backflow, the needle was left in place for 1 minute after injection. Subsequently, the pups were placed on a heating pad and returned to their mothers. Three injections were administered at 12-hour intervals, and the mice were sacrificed 12 hours after the last injection.

[0159]

[0160] (2) Tamoxifen treatment for neonatal Treg markers

[0161] Tamoxifen (Sigma, T5648) was dissolved in corn oil (Sigma, C8267) at a concentration of 5 mg / ml. For neonatal Treg cell lineage tracking, Foxp3eGFP-Cre-ERT2× Rosa26 eYFP Mice were injected intraperitoneally with tamoxifen (35 µg per g body weight) on days 3, 6, and 9 after birth. The mice were then analyzed at 2 or 6 weeks of age.

[0162]

[0163] (3) AAV-mediated increase in Treg

[0164] AAV-PHP.eB vectors expressing mouse Il2 or EGFP driven by the GFAP promoter were constructed using VectorBuilder as previously described. After cloning the mouse interleukin-2 coding sequence (NM_008366.3) or EGFP sequence downstream of the full-length GFAP promoter, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation signal (bGH polyA) were cloned. The vectors were delivered to 5-week-old BTBR mice at a rate of 1 × 10⁶ per mouse. 9 It was administered intravenously at a dose of the vector genome (total volume 100 µL). Animals were analyzed 4 to 5 weeks after injection.

[0165]

[0166] (4) Intravascular immune cell markers

[0167] To label intravascular immune cells, mice were anesthetized with isoflurane and anti-CD45-PE antibody (3 µg per mouse; BioLegend, 103106) was injected posterior to the orbit. Blood was collected after 3 minutes. Neonatal mice (P8) were similarly anesthetized and the antibody was injected into the heart (0.25 µg per mouse). Blood was collected after 1 minute of injection. Then, PBS was perfused through the heart before collecting brain tissue.

[0168]

[0169] 3. Separation of cells from tissue

[0170] Mice were severely anesthetized and perfused through the heart with PBS prior to tissue collection. For brain cell isolation, the brain was mechanically pulverized and enzymatically isolated in DMEM (Welgene) medium containing collagenase IV (1 mg / ml, Gibco), DNase I (0.1 mg / ml, Roche), and 2% FBS (Gibco) at 37°C for 30 minutes with continuous stirring. The pulverized tissue was filtered through a 70 µm cell filter, centrifuged (750 × g, 5 min, 4°C), and resuspended in 35% Percoll (Cytiva) medium dissolved in PBS. Then, the sample was centrifuged (1,120 × g, 20 min, room temperature) to remove myelin debris. To separate the dura mater, the tissue was carefully incised under a microscope, and after enzymatic digestion as described above, it was filtered (70 µm) and centrifuged (750 × g, 5 min, 4 ℃). Lung tissue was finely chopped and separated in DMEM medium containing collagenase IV (3 mg / ml), DNase I (0.3 mg / ml), and 2% FBS at 37 ℃ for 30 minutes, then filtered (40 µm) and centrifuged (750 × g, 5 min, ℃). Red blood cells were lysed using RBC lysis buffer (BioLegend). The colon tissue was epithelialized in RPMI 1640 medium containing 50 mM EDTA and 2% FBS at 37 ℃ for 15 minutes. After removing the Fire patch, the tissue was enzymatically separated in RPMI 1640 medium containing collagenase IV (1.5 mg / ml) and 5% FBS at 37°C for 30 minutes. Cells were filtered through a 70 µm filter and centrifuged at 750 × g for 5 minutes at 4°C. Splenocytes were separated via erythrocyte lysis (RBC lysis buffer; BioLegend) following mechanical disruption using a 40 µm filter. All separated cells were resuspended in RPMI 1640 medium supplemented with 10% FBS prior to further analysis.

[0171]

[0172] 4. Flow cytometry and cell classification of mouse samples

[0173] For flow cytometry analysis, isolated cells were stained with fluorescent conjugated antibodies in PBS. Dead cells were removed using 1:2000 diluted LIVE / DEAD Fixable Aqua (Thermo Fisher Scientific) or Zombie NIR Fixable Viability Kit (BioLegend). For intracellular staining of nuclear proteins, cells were fixed and permeable with Foxp3 / Transcription Factor Staining Buffer (Invitrogen) according to the manufacturer's protocol. For intracellular staining of cytoplasmic proteins, cells were fixed and permeable with Intracellular Fixation and Permeabilization Buffer (Invitrogen) according to the manufacturer's protocol. After staining, cells were washed with PBS, and data were collected using a BD FACSCelesta (BD), Sony ID7000 (Sony), or BD FACSymphony A5 (BD). For cell sorting, isolated cells were stained with fluorescent conjugated antibodies in PBS, washed, and then resuspended in DMEM containing 10% FBS. DAPI was added to distinguish dead cells. Cell classification was performed using a Sony MA900 cell classifier (Sony). Flow cytometry data were analyzed in FlowJo v10.9.0 (BD).

[0174]

[0175] 5. Single-cell RNAseq and TCRseq

[0176] For single-cell analysis of all lymphocytes, live CD45⁺CD11b⁻ lymphocytes and CD45 intCD11b⁺ microglia were isolated from WT mice and mixed in a ratio of approximately 9:1. For single-cell T cell analysis, live CD45⁺TCRβ⁺ T cells were isolated from WT mice. Brain and spleen cells were collected from 10 to 15 mice per group. After isolation, the cells were processed according to the manufacturer's 10X Genomics single-cell sequencing platform user manual. Next-generation sequencing libraries were prepared using the Chromium Next GEM Single Cell 3p RNA Library v3.1 (for lymphocytes), Chromium Next GEM Single Cell 5p RNA Library v2 (for T cells), and Chromium Next GEM Single Cell VDJ Library v2 (for TCRs) according to the manufacturer's instructions (10X Genomics). After sequencing on an Illumina HiSeq X or NovaSeq 6000 platform, data was processed using Cell Ranger v7.0.1 (10X Genomics) to align reads, create a gene expression matrix, and filter the output. The output was then imported into R using the Seurat package for downstream analysis. Quality control filtering was applied to remove cells expressing fewer than 250 unique genes and those with mitochondrial gene expression exceeding 20%. After normalization, principal component analysis and data integration were performed using standard correlation-based methods or Harmony. Subsequently, dimensionality reduction was performed using UMAP, followed by clustering using the FindClusters function. Clusters were manually annotated based on the expression of canonical gene markers. For Treg analysis, Foxp3-expressing cells within CD4⁺ T cell clusters were subset from the T cell sequencing data, and the integration pipeline was applied to this subset. Differentially expressed genes were identified using the FindMarkers function (min.pct = 0.1). Adjusted p < 0.Genes with |Log2FC| > 0.5 and |05 were considered differentially expressed. Gene signature scores were calculated using the AddModuleScore function, and distribution and visualization were evaluated using Nebulosa. The following gene signatures were used for Treg phenotype scores: pan-histological Treg signatures; dormancy, early activation, recovery, and Th1-like signatures (Immgen). The gene sets used for Proliferation (GOBP_CELL_CYCLE) and OXPHOS (KEGG_OXIDATIVE_PHOSPHORYLATION) were retrieved from the MSigDB database. For pathway analysis, differentially expressed genes in each group were analyzed using Metascape. The SCENIC package was used to infer gene regulatory networks within regulatory T cells. Brain Treg scRNA-seq data were integrated with publicly available scRNA-seq datasets from the P7 cortex and hippocampus, and cell-to-cell interaction analysis was performed using CellChat. Principal component analysis (PCA) was performed to visualize transcriptional variation among tissue-derived Treg populations. PCA embeddings were extracted from the integrated dataset, and sample-specific mean coordinates for top principal components were calculated and plotted to show sample-level distribution patterns. scRNA-seq data of VAT, skin, and colon Tregs obtained from previously published studies were included in the analysis. For transcriptome analysis specific to brain Treg cells, cell cycle-related genes were excluded (not shown), and the analysis focused exclusively on 'activated' and 'Th1-like' Treg cell clusters. TCR analysis and visualization were performed using the scRepertoire package, considering only T cells with paired α- and β-chains. Clonal sizes were classified as follows.Clones consisting of a single cell were classified as 'single', clones consisting of two cells as 'small', and clones consisting of three or more cells as 'large'.

[0177]

[0178] 6. Single-nuclear RNAseq

[0179] 6-week-old Foxp3s administered DT or PBS at 8 and 15 weeks of age DTRThe retrosplenial cortex of mice was extracted using the Mouse Brain Matrix (LMS Korea). Tissues collected from three mice per group were gathered for analysis and stored at -80°C until further processing. Single nuclei were isolated from the frozen tissues using the Singulator 100 system (S2 Genomics), followed by the removal of residues using a Percoll concentration gradient. The nuclei were then processed according to the manufacturer's instructions for the Chromium Next GEM Single Cell 3′ RNA Library Kit v3 (10x Genomics). After sequencing on the Illumina HiSeq X platform, the data were processed using Cell ranger v7.0.1 (10X Genomics). The results were then imported into R using the Seurat package, and subsequent analyses were performed as described above. To ensure that only retrosplenial cortex cells were analyzed, the dataset was integrated with the Allen Brain Map single-cell RNA-seq reference atlas of the mouse cortex and hippocampus. Cells were filtered based on region annotations, excluding only clustered cells labeled 'RSP' (post-ampullary cortex) from the reference dataset. Cell type annotations were created by referencing cell types annotated in the Allen Brain Map scRNA-seq database. Statistical analysis of within-cluster proportions for each cluster was performed using scProportionTest. The following gene signatures were used for glial cell scoring: inflammatory astrocytes, mature and immature astrocytes, and disease-associated microglia. The gene sets used for IL-4 response (GSE16385_UNTREATED_VS_12H_IL4_TREATED_MACROPHAGE_UP) and IFNγ response (HALLMARK_INTERFERON_GAMMA_RESPONSE) were retrieved from the MSigDB database.A set of genes related to Alzheimer's disease and Parkinson's disease was extracted from RNA-seq data of postmortem brain tissue and represents genes upregulated (FC > 1.5, FDR < 0.05) in the patient's brain.

[0180]

[0181] 7. In vitro analysis

[0182] (1) Ex vivo stimulation of T cells

[0183] T cells were isolated from the brains and spleens of 4–5 week old mice that either suffered from Treg deficiency during the neonatal period or received PBS as a control. The isolated cells were cultured in T cell medium at a density of 20,000 cells per well in round-bottomed 96-well plates supplemented with Dynabeads™ Mouse T-Activator CD3 / CD28 (Invitrogen) for 72 hours, after which the culture supernatant was collected. For cytokine detection via flow cytometry, immune cells were isolated from the brain, cultured in T cell medium, and then stimulated for 4 hours in a 37°C, 5% CO2 incubator with the addition of eBioscience™ Cell Stimulation Cocktail and a protein transport inhibitor (Invitrogen). Subsequently, intracellular cytokine staining was performed as described above.

[0184]

[0185] (2) Microglia synaptosome engulfment assay

[0186] Synaptosomes were prepared from the brains of adult C57BL / 6J mice using the Syn-PER synaptic protein extraction reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The resulting synaptosome suspension was stored at -80 °C in Syn-PER reagent supplemented with 5% (v / v) DMSO. Conjugation with pHrodo dye was performed as previously described. Briefly, synaptosomes at a concentration of 1 mg / mL were incubated with 12 µg of pHrodo iFL Green STP Ester (amine-reactive; Thermo Fisher Scientific, P36012) in 100 mM sodium bicarbonate buffer (pH 8.3) at room temperature for 1 hour while rotating in the dark. After labeling, synaptosomes were centrifuged at 15,000 × g for 20 minutes at 4 ℃ to pellet the samples, washed once with PBS to remove unbound dye, and then resuspended in Syn-PER reagent containing 5% (v / v) DMSO. The final culture medium was stored at -80 ℃ for long-term use.

[0187] Mixed glial cell culture medium was enzymatically digested from the cortex of mice 1 to 2 days old using 0.125% trypsin-EDTA (Invitrogen) for 5 minutes and then completely separated by pipetting. After short centrifugation, the cells were resuspended in DMEM / F12 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco) and cultured for more than 2 weeks. After the culture period, cells were separated using 0.125% trypsin-EDTA, and cell sorting was performed to separate microglia.

[0188] For cytokine treatment, microglia were cultured in DMEM / F12 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco) and treated with mouse recombinant IFNγ (R&D Systems, 485-MI-100), IL-4 (Peprotech, 214-14), IL-5 (R&D Systems, 405-ML-005), and IL-13 (R&D Systems, 413-ML-025) at a concentration of 50 ng / mL, or with all cytokines in combination. PBS was used as a control. The culture medium was maintained for 24 hours, and synaptosomes labeled with pHrodo were added at a final concentration of 50 µg / mL one hour before cell collection. For the co-culture experiment, T cells isolated from the brains of DT-injected pups and spleens from control pups injected with PBS (as described above) were co-cultured with 14,000 microglia in a round-bottomed 96-well plate with 1 µg / mL of anti-mouse CD3ε (BioXCell, BE0001-1) antibody to achieve a T cell-to-microglia ratio of 1:2. A mixture of 10 µg / mL of anti-IFNγ (BioXCell, BE0055) antibody and 10 µg / mL of anti-IL-4 (BioXCell, BE0045) antibody, or the corresponding isomorphic control (BioXCell, BE0088), was added to the culture medium. Co-culture was maintained for 48 hours, and synaptosomes were added at 50 µg / mL one hour prior to cell collection. After staining with a surface marker, the cells were analyzed using ID7000 (Sony).

[0189]

[0190] 8. Multi-cytokine analysis

[0191] For multi-cytokine analysis using culture supernatant, cytokine levels were measured using the LEGENDplex™ Mouse Th Cytokine Panel (BioLegend, 741044) and Mouse Cytokine Panel 2 (BioLegend, 740134). For multi-chemokine analysis using brain tissue lysate, the posterior ampullary cortex was incised using mouse brain stroma (LMS Korea), homogenized with radio-immunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific), and lysed by sonication. Chemokine levels were measured using the LEGENDplex™ Mouse Inflammatory Chemokine Panel (BioLegend, 740007) and Mouse Inflammatory Chemokine Panel 2 (BioLegend, 741068). For each sample, over 300 beads were collected using an ID7000 flow cytometer (Sony) and analyzed using the cloud-based platform LEGENDplex data analysis software suite (legendplex.qognit.com / ). Chemokine concentrations were normalized to total protein content and quantified using the Pierce™ BCA protein analysis kit (Thermo Fisher Scientific).

[0192]

[0193] 9. Neuropixels recording

[0194] All mice were tested at least 8 weeks of age. In vivo recordings were performed under urethane anesthesia (1.5 g / kg) with the mice's heads secured in a stereotactic device. Body temperature was monitored and maintained using a thermostat (CWE, TC-1000). Vaseline was applied to the mice's eyes to prevent corneal damage during the procedure. NeuroPixel Probe 2.0 was inserted unilaterally at coordinates AP -2.42–3.22 mm, ML ± 0.3 mm (bregma), and brain surface -1.50 mm (posterior ependymal cortex) to target the posterior ependymal cortex. The probes were coated with the fluorescent dye DiI (Thermo Fisher Scientific, D7757) to identify the recording site. The location of each probe channel was determined by examining post-processed brain tissue from each mouse using Allen-CCF (github.com / cortex-lab / allenCCF).

[0195] The recordings used for analysis were performed for 45 minutes after stabilization for 30 minutes following insertion. The probe was inserted at a speed of 20 µm per minute using an MPM system micromanipulator (New Scale Technologies). Simultaneous recordings were taken at 384 sites across four shanks of the Neuropixels probe 2.0. Electrophysiological data were collected using SpikeGLX (billkarsh.github.io / SpikeGLX / ). To obtain single-unit data, electrode signals were filtered between 300 Hz and 6,000 Hz and sampled at 30,000 Hz. Spike classification was performed using Kilosort 1.5 (github.com / MouseLand / Kilosort / ), after which separated single units were manually screened using Phy (github.com / cortex-lab / phy). Single units were selected through a comprehensive examination based on established metrics such as inter-spike intervals (ISI), autocorrelation, and waveform morphology. Then, single-unit data was analyzed using custom MATLAB code to calculate the firing rate. Burst firing was defined as a sequence of two or more consecutive spikes with an interval between spikes of less than 10 ms, and was quantified as the ratio of the total spikes occurring within that burst to the spikes obtained during the entire recording period.

[0196] Local field potential (LFP) data (sampling rate: 2000 Hz) was band-pass filtered between 0.1 Hz and 200 Hz using a third-order Butterworth filter, and power line noise was attenuated by applying a notch filter. The filtered LFP signals were analyzed using a custom MATLAB script. The power spectrum of each electrode channel was calculated using the Fourier Transform. This was achieved by dividing the data into 30-second intervals and applying the Fast Fourier Transform (FFT) to each bin. The power spectrum was normalized by dividing the power at each frequency by the total power of all frequencies. The frequency bands of interest were defined as delta (0.1–4 Hz), theta (4–12 Hz), beta (13–30 Hz), low-gamma (31–55 Hz), and high-gamma (65–130 Hz). For each electrode channel, the power of each frequency band was calculated by summing the normalized power spectra within the defined band limits. The power values ​​of each frequency band and brain region were log-transformed to facilitate analysis.

[0197]

[0198] 10. Immunohistochemical staining

[0199] For immunohistochemical staining, the brain was perfused with PBS and fixed by adding 4% PFA to the PBS. Then, equilibration was achieved by adding 30% sucrose to the PBS, and after freezing in OCT solution (Sakura Finetech), sections were prepared to a thickness of 20 or 50 µm using a cryostat. The sections were placed in a blocking solution (PBS containing 5% normal donkey or goat serum and 0.3% Triton X-100) and blocked at room temperature for 1 hour. These were incubated overnight at 4°C in blocking solution with the following primary antibodies: rabbit monoclonal anti-CD3 (1:100, ab16669, Abcam), rat monoclonal anti-CD45 (1:200, 14-0451-82, Invitrogen), Armenian hamster monoclonal anti-CD31 (1:100, MA3105, Invitrogen), chicken polyclonal anti-GFP (1:200, GFP-1020; Aves Labs), rabbit polyclonal anti-IBA1 (1:500, 019–19741; Wako), goat polyclonal anti-IBA1 (1:200, ab5076, Abcam), rabbit polyclonal anti-PSD-95 (1:200, 51-6900; Invitrogen). Sections were washed and incubated with an appropriate secondary antibody (1:500) at room temperature for 2 hours or overnight at 4°C, after which they were enclosed in mounting medium containing DAPI. Tile scan images were obtained using a THUNDER imager (Leica) and LAS X software (Leica). Confocal microscopy images were obtained using an LSM700 (Carl Zeiss) equipped with a 63× / 1.40 oil objective lens or an FV1000 (Olympus) equipped with 60× / 1.35 oil and 100× / 1.40 oil objective lenses. For quantitative analysis, acquisition parameters including laser power, pinhole size, gain, and offset were kept constant across all experimental groups.

[0200]

[0201] 11. Video Analysis

[0202] (1) Microglial morphology analysis

[0203] For the analysis of microglia morphology, a total of 20–25 optical sections were acquired at 1 µm intervals from the retrosplenial cortex region of 4-week-old neonatal Treg-deficient mice and control mice using a 60x objective lens (800 × 800 pixels; 212 × 212 µm). Microglia morphology was reconstructed using the semi-automatic 3DMorph plugin. Skeletal images were analyzed using the ImageJ Sholl Analysis plugin.

[0204]

[0205] (2) Quantification of PSD-95 Punta in microglia

[0206] To quantify PSD-95 puncta within microglia, more than 35 optical sections were acquired from the posterior ampulla of 4-week-old neonatal Treg-deficient mice and their collateral controls using a 100x objective lens (1024 × 1024 pixels; 126.98 × 126.98 μm) with a 1-μm z-step. Image processing was performed in ImageJ. Background noise in the Iba1 channel was removed using the "Subtract Background" function with a rolling ball radius of 25 pixels, followed by the application of Gaussian blurring with a sigma value of 1. Iba1-positive microglia were segmented using the Otsu method with an automatic threshold, and a binary mask was generated. PSD-95 signals within the Iba1-positive region were separated using the "Image Calculator" function. The number of PSD-95 puntas in each Iba1-positive microglia was quantified using the "find maximum" function with a prominent threshold set to 35.

[0207]

[0208] (3) PSD-95 density quantification

[0209] To quantify PSD-95 positive synaptic sites, regions within the posterior ampulla exhibiting CD45⁺ immune cell infiltration were identified by visual inspection. Images of the infiltration area and adjacent regions within the same cortical layer were acquired at 2 µm intervals using a 100x objective lens (1024 × 1024 pixels, 126.98 × 126.98 µm). To ensure signal consistency, site quantification was performed on six optical sections per sample at depths of 10–20 µm below the surface. The number of PSD-95 sites was quantified using ImageJ's "Find Maxima" function with a salience threshold set to 400.

[0210]

[0211] (4) Whole-brain mapping of immune cell infiltration

[0212] Sagittal sections were collected at 200 µm intervals from one hemisphere of the brain of 4-week-old neonatal Treg-deficient mice. Tile scan images of DAPI and CD45 staining were acquired using a THUNDER Imager (Leica) equipped with a 20x objective lens. Spatial registration for the Allen Mouse Brain Atlas (CCFv3) was performed using the QUINT workflow. DAPI signals were used for initial alignment in QuickNII and subsequently fine-tuned manually using VisuAlign. The resulting output files were processed in Nutil, and the number of objects and signal density within the brain regions defined in the Allen Mouse Brain Atlas were calculated using binary masks of the CD45⁺ signals. Whole-brain 3D visualizations of CD45⁺ immune cell distribution were generated using the MeshView application.

[0213]

[0214] 12. Behavioral Evaluation

[0215] Behavioral tests were conducted on male and female mice aged 8 weeks or older under ambient lighting conditions of 20–30 lux. Mice underwent only one behavioral test during the lighting cycle (12:00–16:00). All mice were tested after 8 weeks of age, and age- and sex-matched mice were used as a control group. Mice were moved to the testing area at least one hour prior to the test to acclimatize. All equipment and testing areas were wiped with 75% ethanol between each session and test to ensure no residual odors remained.

[0216]

[0217] (1) Open field test

[0218] Rats were placed in an open space (45 x 45 x 35 cm) and allowed to explore freely for 10 minutes. During the experiment, tracking software EthoVision XT 14 (Noldus) was used to monitor the rats' movements and analyze the distance traveled, speed, and time spent in the center of the experimental area. The central area was defined as an inner 22 x 22 cm area.

[0219]

[0220] (2) Novel object / location recognition (NOR / NLR) task

[0221] The behavioral paradigm adopted a previously established new object recognition protocol. This task was performed for 24 to 48 hours after the open experiment, which was also used as a habituation phase. Mice were instructed to explore a square open experimental area (45×45×40 cm) with conspicuous spatial signals (black circular markers) attached to one wall of the device.

[0222] During the familiarization phase, mice were exposed to two identical objects for 10 minutes and then returned to their original cages. Two objects of the same height and material (cylindrical or square pillars) were used to be positioned within 3 cm of the signal. Object placement was randomized to control for the animals' innate preferences. To ensure a sufficient first attempt to familiarize the mice with the objects, this session was repeated after 15 minutes. Two hours after the familiarization session, one of the familiar objects was replaced with a new object, and the mice were reintroduced into the enclosure for 10 minutes (NOR test). Object navigation was defined as the mouse turning its head within 2 cm of the object and actively sniffing it. In the New Localization (NLR) test, one of the familiar objects was moved to a new location 20 cm away from its original position and placed on the opposite side of the spatial signal. This test was performed 15 minutes after the NOR test, and the mice were allowed to navigate for another 10 minutes. All sessions were videotaped, and the time taken to navigate each object was quantified by two independent, blinded observers.

[0223]

[0224] (3) Y-maze test

[0225] To evaluate spatial working memory, a Y-maze voluntary alternation test was administered according to an existing protocol. This test consisted of three identical arms (6 × 30 × 15 cm, A, B, C), with each arm positioned at a 120° angle to the others. Mice were placed in the center of the maze and allowed to explore freely for 10 minutes. An arm entry was defined as when all four paws entered an arm area. Voluntary alternation behavior was defined as entering all three arms consecutively without returning to an arm previously entered. The alternation rate was calculated using the following formula: (Number of voluntary alternations / (Total number of entries - 2)) × 100%.

[0226]

[0227] (4) Repetitive behavior test

[0228] Compulsive-like behaviors were evaluated by measuring self-grooming and digging activities in a new environment, as previously described. Each mouse was placed in a new home cage filled with new bedding and acclimatized for one minute. Behaviors were recorded for ten minutes. Self-grooming was defined as licking or scratching the face or body, and digging behavior was defined as moving the bedding using the forelimbs or snout. Repetitive behaviors were scored manually. All scoring was performed by two independent observers who were unaware of the experimental conditions.

[0229]

[0230] (5) Interpersonal Social Interaction Test

[0231] To evaluate intersocial behavior, two age- and sex-matched stranger mice were placed together in a clean cage (39 × 20 × 16 cm) without bedding. After 15 minutes of interaction, the mice were returned to their original cages. Video recordings were analyzed in a blinded manner by independent observers, and direct social behaviors such as nose-to-nose, nose-to-body odor sniffing, close following, grooming, physical contact, and crawling over or under the partner mouse were quantified.

[0232]

[0233] (6) 3-chamber test

[0234] Sociality was evaluated using a 3-chamber apparatus according to the previously described method. The experimental area (64 × 35 × 35 cm) was divided into three interconnected rooms of equal size with removable doors between the partitions. A wire mesh cage (cylindrical metal cup) was placed at one end of each lateral room. An area within a radius of 3 cm centered on each cup was defined as the interaction zone. During the habituation process, the subject mice were placed in the center of the three rooms and allowed to freely explore the other rooms for 5 minutes. After habituation, the mice were immediately placed back in the center. Unfamiliar mice of the same species, matched by sex and age, were placed in one wire mesh cage, while an inanimate object of similar size and shape was placed in another cage. The positions of social and object stimuli were alternated in each experiment to regulate lateral preference. The mice were allowed to freely explore all three rooms for 10 minutes. The time spent in each interaction zone was recorded. The sociality index was calculated by dividing the time spent in the social zone by the total time spent in both zones.

[0235]

[0236] (7) Stress sensitivity test

[0237] A protocol modified based on previous reports was used to assess vulnerability to stress-induced social impairment. Mice were subjected to restraint stress for 4 hours daily during a 7-day continuous period of light exposure. This paradigm was selected because it does not induce obvious abnormalities in social behavior. The 3-chamber social interaction test described above was administered before and after the 7-day stress exposure to evaluate changes in social interaction behavior.

[0238]

[0239] 13. Notice on Human Research Ethics

[0240] The PBMC immune profiling study was approved by the Institutional Review Board (IRB) of Severance Hospital, Yonsei University College of Medicine (4-2019-0926). Written consent was obtained from all participants and their legal guardians prior to the start of the study. The clinical trial investigating LdIL-2 treatment in patients with ASD was an open-label, single-arm study without randomization. This clinical trial was conducted at the Second Affiliated Hospital of Kunming Medical University after receiving approval from the Institutional Review Board and registering in the Chinese Clinical Trials Registry (ChiCTR2000040836; chictr.org.cn / ).

[0241]

[0242] 14. Selection of Samples for Research Cohorts and Human Studies

[0243] (1) Peripheral blood mononuclear cell (PBMC) immune profiling study

[0244] Blood samples were collected from 76 children with Autism Spectrum Disorder (ASD) and 43 typically developing children (TDC). Children with ASD met the following inclusion and exclusion criteria: 1) children aged 4 to 15 years who were diagnosed by a child and adolescent psychiatrist according to the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition; 2) children whose diagnosis was supplemented by the Autism Diagnostic Observation Schedule-2 (ADOS-2), the Autism Diagnostic Interview-Revised (ADI-R), and the Social Responsiveness Scale (SRS); 3) Children with mental disorders other than autism were excluded based on semi-structured psychiatric interviews and the Kiddie-Schedule for Affective Disorders and Schizophrenia-Present and Lifetime Version (K-SADS-PL). The TDC group was recruited through in-hospital bulletin boards and external advertisements. Participants in the TDC group met the following inclusion and exclusion criteria: 1) being between 4 and 15 years of age and exhibiting typical developmental patterns with a full-scale intelligence quotient (FSIQ) of 80 or higher; 2) being screened for Autism Spectrum Disorder (ASD) characteristics using the SRS; and 3) children with a history of mental disorders were excluded based on semi-structured psychiatric interviews and parental reports.

[0245]

[0246] (2) LdIL-2 clinical trial

[0247] The inclusion criteria for all participants are as follows: 1) Ages 3–16. 2) Peripheral immune abnormalities (one of the following): ① Treg cell ratio ≤ 2.21%. ② Th1 cell ratio ≥ 2.44%. ③ Th2 cell ratio ≥ 0.73%. ④ Th17 cell ratio ≥ 0.52%. ⑤ Tc1 cell ratio ≥ 7.39%. ⑥ Th1 / Treg cell ratio ≥ 0.63. ⑦ Th2 / Treg cell ratio ≥ 0.17. ⑧ Cytokines TNF-α ≥ 2.49 pg / ml; IL-4 ≥ 5.29 pg / ml; IL-5 ≥ 2.81 pg / ml; IL-17A ≥ 3.05 pg / ml. ⑨ Positive detection of anti-cerebrovascular endothelial cell antibodies in peripheral blood. (3) No immunological treatment in the past 2 weeks. (4) Diagnosis of autism spectrum disorder (ASD) according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5).

[0248] Exclusion criteria are as follows: 1) History of other neurological or psychiatric complications such as seizures, schizophrenia, or substance abuse. 2) Previous related treatment. 3) Use of prescribed antibiotics, probiotic products, or immunomodulators within the past 2 weeks. 4) Allergy to IL-2 or similar products, or allergic constitution. (5) Abnormal liver and kidney function or liver and kidney disease. 6) Hematological disorders. 7) Organic diseases. 8) Other conditions deemed inappropriate by the investigator for participation in the study.

[0249] During the treatment period, each participant received LdIL-2 therapy. Based on previous studies, a dose of 16,000 IU / kg of IL-2 was administered via subcutaneous injection every other day for two weeks. This regimen consisted of seven injections followed by a two-week rest period, constituting a single treatment cycle lasting a total of four weeks. All patients received treatment for an initial 12 weeks, which included three treatment cycles using IL-2. Subsequently, they underwent an additional 12 weeks of follow-up without the study medication. The parents or legal guardians of all participants provided written informed consent. Three of the participants demonstrated good outcomes after completing the three treatment courses. With parental consent, they received three additional treatments, bringing the total treatment duration to six months.

[0250] The primary outcome measured was changes in autistic behavior assessed using the Childhood Autism Rating Scale (CARS) and the Autism Treatment Evaluation Checklist (ATEC) after a 3- or 6-month period. The CARS ranges in total score from 15 to 60 points based on the sum of 15 items, with higher scores indicating more severe autism. In contrast, the ATEC consists of a total of 77 items and includes four subscales: expressive / verbal communication, social competence, perceptual / cognitive ability, and health / physiological / behavioral. Additional outcomes included assessments using the Abnormal Behavior Checklist (ABC), Caregiver Strain Questionnaire (CGSQ), Hospital Anxiety and Depression Scale (HAD), and Autism Mental Status Exam (AMSE). Regarding immunological outcomes, this study evaluated changes in peripheral blood lymphocyte subsets and cytokine levels from baseline to the 3- or 6-month mark. Safety outcomes included monitoring for adverse events such as injection site reactions, infections, upper respiratory infections, and fever. Simultaneously, patients' biochemical indicators were closely monitored.

[0251]

[0252] 15. Flow cytometry of human specimens

[0253] For the immune profiling of ASD patients, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque PLUS (Cytiva) density gradient centrifugation. Briefly, whole blood was diluted 1:1 with PBS, Ficoll solution was placed in a conical tube, and centrifuged at 1,100 × g for 20 minutes at 23 °C in a brake-free swing bucket rotor. The mononuclear cell layer was collected, diluted in 20 mL of PBS, and centrifuged at 700 × g for 10 minutes at 23 °C. Subsequently, 2 × 10⁶ PBMCs were placed in a frozen medium containing 70% fetal bovine serum (Gibco), 20% RPMI-1640 (Welgene), and 10% DMSO (Sigma-Aldrich). 6 The cells were resuspended at a cell / mL concentration and stored at -80°C. The frozen PBMCs were thawed and recovered frozen prior to analysis. Half of the recovered cells were cultured in 96-well round-bottom plates containing T cell medium and stimulated for 4 hours at 37°C (5% CO2) using the eBioscience™ Cell Stimulation Cocktail and a protein transport inhibitor (Invitrogen). The remaining cells were cultured without stimulation for 3 hours under the same conditions in the same medium. For surface marker analysis, cells were stained at 4°C for 30 minutes. For intracellular protein detection, cells were fixed and permeable using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen), followed by intracellular staining for 40 minutes at room temperature. To evaluate cytokine production, stimulated cells were washed with PBS and surface stained; after treatment with the eBioscience™ Intracellular Fixation and Permeation Buffer (Invitrogen), intracellular cytokine staining was performed for 40 minutes at room temperature. The sample was washed and analyzed using BD FACSCelesta (BD Biosciences).

[0254] For the analysis of patients treated with LdIL-2, peripheral blood samples were collected before and after treatment using EDTA anticoagulant tubes (3 mL) and serum separation tubes (5 mL) (BD Biosciences). Plasma was separated by centrifuging the EDTA-treated blood at 1,000 × g for 10 minutes and stored at -80 °C for future analysis. Peripheral blood monocytes (PBMCs) were isolated using SepMate™ tubes containing lymphocyte isolation medium (STEMCELL Technologies) for subsequent flow cytometry analysis. The isolated PBMCs were washed with PBS and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin / streptomycin. Cells were stimulated with PMA / ionomycin and GolgiStop™ protein transport inhibitor (BD Biosciences) for 5 hours in a 37 °C, 5% CO2 incubator. Staining, fixation, and permeation were performed using the Human Th1 / Th2 / Th17 Phenotyping Kit from BD Biosciences according to the manufacturer's instructions. Samples were washed and analyzed using the DX Flex (Beckman Coulter).

[0255]

[0256] 16. Cytokine analysis of human specimens

[0257] Serum samples were collected by centrifugation at 2,000 × g for 5 minutes and stored at -80 °C for cytokine analysis. Serum cytokine concentrations were quantified using the Human 14-plex Kit (QuantoBio, C60011). Active TGF-β concentrations were quantified using the Human TGF-β1 1-plex Kit (QuantoBio, B111206) according to the manufacturer's standard protocol, including the acidification step required for the detection of the active form. Briefly, antibody-conjugated beads were added to a 96-well plate and incubated with serum or plasma samples and standards at room temperature with gentle shaking. After washing, biotinylated detection antibodies were added, and the plates were incubated at room temperature. Then, streptavidin-PE was applied, followed by additional washing. After adding the final buffer, data were collected using the DX Flex (Beckman Coulter). Cytokine concentrations were calculated using FCAP Array™ Software v3.0 (BD Biosciences).

[0258]

[0259] 17. Statistical Methods

[0260] Bar graphs and dot plots represent the means, and error bars represent the standard error of the means. Group comparisons were performed using GraphPad Prism 10 (GraphPad Software) or R version 4.1.3 (The R Foundation). Two-sided unpaired Student's t-tests were performed for comparisons between two independent groups. For gene signature score analysis, the Mann-Whitney U test was used for comparisons between two independent groups, while the Kruskal-Wallis test with Dunn's multiple comparisons was used for three or more groups. For the Neuropixel experiment, neuronal firing rates and burst rates between groups were compared using the Kolmogorov-Smirnov test, and local field potentials were analyzed using the Mann-Whitney U test. One-way ANOVA with appropriate multiple comparison tests was used for comparisons involving three or more independent groups. For experiments using two factors, two-way ANOVA with repeated measures and appropriate multiple comparison tests was used. Pairwise analysis was performed on samples drawn from the same subjects. A mixed-effects model applying Tukey's multiple comparison test was used to analyze pre- and post-treatment differences in ASD clinical data. Spearman correlation analysis was performed using the rcorr function from the Hmisc package (v5.0.1) in R version 4.1.3. Statistical significance levels were defined as follows: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****).

[0261]

[0262] [Experimental Results]

[0263] 1. Single-cell map of the brain immune environment throughout the developmental process

[0264] Although various immune cell subpopulations affecting brain function have been identified in recent studies, the developmental dynamics and functional characteristics of these cells within the brain are not yet well understood. In this invention, the presence of CD45+ immune cells in the brain parenchyma, lateral ventricles, perivascular space, and choroid plexus was confirmed for the first time during the embryonic (E16), neonatal (P8), and pediatric (P28) periods (Fig. 1a). To comprehensively analyze these cells, CD45+ immune cell populations were isolated along with microglia from the brains of E16.5, P8, and P28 mice, and age-matched fetal livers and spleens were isolated together to perform single-cell RNA sequencing (scRNA-seq) (Fig. 1b). Unbiased clustering analysis revealed various immune subpopulations, including B cells, αβ T cells, γδ T cells, natural killer (NK) cells, and ILCs (Figs. 1c, d). Among these, B cells were the most abundant at all developmental stages, γδ T cells and NK cells were already present during the embryonic stage, and αβ T cells, including CD4+ T cells, appeared mainly after birth (Fig. 1e, f). Flow cytometry analysis across eight developmental stages (E16–adult) confirmed the validity of these findings. The early appearance of γδ T cells and NK cells, particularly γδ T cells, peaked at P28, while αβ T cells gradually increased (Fig. 1g).

[0265] Considering that γδ T cells are significantly distributed at all developmental stages (Figs. 1f, g), the present invention analyzed γδ T cells as a representative subgroup of brain-specific immune specialization. Uniform manifold approximation and projection (UMAP) clustering identified distinct fetal γδ T cell subgroups, clearly distinguishing between pure cells and γδT17 cell clusters. In particular, γδT17 cells were significantly more abundant in the embryonic brain compared to the fetal liver. This is consistent with recent observations of specific early growth of central nervous system-related γδT17 populations (not shown). While embryonic γδ T cells primarily express Il22, postnatal brain γδ T cells highly express Il17a, demonstrating that functional polarization is distinctly observed according to developmental stage (not shown). Differential expression analysis revealed that IL-17 pathway genes (e.g., Rora, Il23r) and early activation markers (Jun, Fos) were significantly more abundant in brain γδ T cells compared to peripheral lymphoid cells (not shown). Furthermore, developmental stage-specific transcriptional profiles were clearly evident. E16 brain γδ T cells preferentially expressed genes related to the cell cycle and migration, P8 γδ T cells showed increased expression of inflammatory cytokines, and P28 γδ T cells exhibited characteristics of mature leukocyte activation, demonstrating dynamic functional changes across developmental stages (not shown).

[0266] Further single-cell and flow cytometry analysis revealed a transient but significant increase in regulatory T cells (Tregs) specifically in the brains of newborns (P8), which is consistent with the dynamic functional specialization of the early lymphocyte population (Fig. 1f). In particular, scRNA-seq analysis confirmed a distinct and transient increase in Foxp3+ Ikzf2+ Tregs specifically in the newborn brain, which clearly exceeded the frequency observed in lymphoid organs (Fig. 1h). Confocal microscopy of Foxp3-EGFP reporter mice confirmed the anatomical location of Tregs within the brain parenchyma, choroid plexus, and the cisternal compartment of the subarachnoid space, distinctly separated from the CD31+ vascular system (Fig. 1i). Flow cytometry further validated these observations, showing that brain Treg frequency peaked significantly at approximately 20% of total CD4+ T cells at 8 months of age (P8), which was significantly higher than the rates observed in other tissues, and remained stable into adulthood (Fig. 1j-l). To directly assess the presence and persistence of Tregs in the brain, Foxp3eGFP-Cre-ERT2× Rosa26 eYFPLineage tracking experiments were performed on reporter mice. Treg cells labeled early in life showed remarkable stability in brain tissue and maintained a retention rate of approximately 60% even at 6 weeks post-labeling. In contrast, labeled T cells rapidly decreased in peripheral lymphoid and non-lymphoid tissues (not shown). A comparative analysis of T cell presence between the dura mater and the deep compartments of the central nervous system revealed that T cell abundance in the neonatal dura mater was negligible at 8 months of age (P8) and increased significantly only upon entering adulthood. Conversely, the number of brain T cells remained constant from the neonatal period to adulthood (not shown). This suggests that T cells initially migrated to the deep compartments of the central nervous system and then moved outward toward the meninges during early postnatal development. Collectively, these experimental results provide a dynamic single-cell map of brain-resident immune cells throughout development and demonstrate that neonatal brain T cells are potential regulators of central nervous system immune homeostasis and neurodevelopment.

[0267]

[0268] 2. Early Development and Specialization of Brain-Residential T Cells (Tregs)

[0269] Tissue-resident T cells possess a unique transcriptional program that performs roles specialized in tissue-specific immune homeostasis and organ function. However, the developmental characteristics of T cells in the central nervous system have not yet been clearly elucidated. To address this issue, single-cell scRNA-seq and T cell receptor sequencing (TCR-seq) were performed on TCRβ+ T cells isolated from the brains and spleens of 1-week-old (1W) and adult (12W) mice (Fig. 2a). Through unsupervised clustering, five transcriptionally distinct T cell subpopulations were identified: "stationary," "active," "TH1-like," "proliferative," and "recirculating" (Fig. 2b, c). Splenic T cells predominantly comprised the "stationary" subpopulation, characterized by high expression of Bcl2, Ccr7, and Klf2, which reflects typical lymphocyte resident and tissue efflux characteristics (Fig. 2b-d). In contrast, brain Tregs abundant in the "activating" and "TH1-like" subgroups highly expressed immunomodulatory markers (Tnfrsf4, Tigit), tissue resident markers (Cd69), and TH1-related genes (Cxcr3, Gzmb, Klrg1) (Figs. 2b-d). A unique "proliferative" subgroup, specifically abundant in the 1W of the brain, significantly expressed cell cycle-related genes (Nusap1, Cdk1), exhibiting active focal expansion during early life (Figs. 2b-d). Conversely, "recycling" Tregs, defined by migration-related genes (Dock2), were primarily expressed in the spleen (Figs. 2b, c). Analysis by overlaying selected gene signature scores onto UMAP plots confirmed the unique characteristics of each subgroup and particularly highlighted the co-expression of tissue resident and proliferative signatures in neonatal brain Tregs (Fig. 2e).Comparative transcriptional profiling of brain Tregs and spleen Tregs confirmed that tissue adaptation genes (Rora, Nfil3), early activation genes (Junb, Nr4a1), and immunomodulatory markers (Ctla4, Gzmb, Icos) were brain-specifically abundant at both stages, suggesting stable tissue adaptation from early infancy (not shown). Principal component analysis (PCA) confirmed that neonatal brain Tregs were distinctly different from Tregs derived from other tissues, which is mainly attributed to proliferation-related characteristics, whereas adult brain Tregs exhibited transcriptional profiles distinct from lymphatic and non-lymphatic tissue subgroups (Fig. 2f).

[0270] Time analysis comparing neonatal (1W) and adult (12W) brain Tregs revealed developmental changes in functional specialization. Neonatal brain Treg cells were distinctly involved in oxidative phosphorylation (OXPHOS) and migration pathways, whereas adult brain Treg cells showed increased signaling via IFNγ and SMAD pathways, indicating a mature and potentially inflammatory responsive state (Fig. 2g-i). Gene signature analysis confirmed that proliferative and metabolic gene expression increased early in life, transitioning into mature tissue resident markers by adulthood (Fig. 2j).

[0271] To further investigate the upstream regulatory mechanisms of brain Treg cell specialization, regulon analysis confirmed that the transcription factor Cebpb is specifically abundant only in neonatal brain Treg cells, a finding confirmed by increased protein expression measured via flow cytometry. This suggests a unique regulatory program that shapes the brain-specific Treg cell phenotype (not shown). Beyond transcriptional regulation, the integration of single-cell transcriptome data from the neonatal brain population revealed the existence of various potential interactions between brain regulatory T cells and neurons, glial cells, and endothelial cells via signaling pathways such as semaphorin–plexin. This suggests potential mechanisms by which brain regulatory T cells regulate central nervous system development and function (not shown). In addition to regulatory T cells, brain-resident CD4+ conventional T cells (Tconv) and CD8+ cytolytic T cells (CTLs) also exhibited transcriptional patterns different from those of spleen-resident T cells, suggesting more extensive and tissue-specific immune specialization within the brain. Brain Tconv were rich in activated receptors and cytokine-mediated signaling pathways, and brain CTLs specifically upregulated interferon, TNF, and MAPK signaling genes. This stands in stark contrast to splenic T cells, which primarily express genes related to ribosome biosynthesis and methylation (not shown). These results demonstrate that brain-resident T cells, extending beyond Treg cells, adapt unique functional characteristics specialized for central nervous system immune regulation.

[0272] Next, standard Treg functional markers were validated at the protein level using flow cytometry. Neonatal brain Tregs showed significantly high expression of proliferation (Ki-67), activation (ICOS), and brain-specific Th1 markers (T-BET), indicating an early activation and proliferation state that occurs only during the neonatal period (Fig. 1k). In contrast, adult brain Tregs showed a gradual increase in the expression of mature tissue-resident markers CD103 and CD69, as well as increased CTLA-4 expression. This suggests that stable tissue resident and immune-modulatory functions were gradually acquired over time (Fig. 1k). In particular, T-BET expression specifically increased in 1W brain Tregs, suggesting specialization that occurs during the early growth phase, distinct from peripheral Tregs (Fig. 1k). These findings suggest that brain-resident T cells gradually mature and acquire stable tissue adaptation functions throughout the development of the central nervous system after birth. Furthermore, LGALS3, known for its regulatory effects on central nervous system glial cells and endothelial cells, was selectively and abundantly expressed in neonatal brain T cells (Fig. 1k). Comparative analysis between cerebral circulating T cells and peripheral circulating T cells revealed particularly high expression of ICOS, CTLA-4, and Ki-67 in neonatal brain T cells, further supporting their specific phenotype (not shown). Intracardiac labeling using anti-CD45-PE antibodies was performed to rule out potential contamination from circulating cells, and approximately 20–30% of the brain T cells were confirmed to be PE-positive (not shown). Notably, these PE-positive T cells maintained high expression of ICOS, CTLA-4, and Ki-67, strongly suggesting that they are transitional cells actively adapting to the central nervous system microenvironment, rather than passively recruited from the circulatory system as previously suggested. These data collectively support the view that brain-resident T cells exhibit distinct functional specialization and gradual tissue adaptation during the early postnatal development of the central nervous system.

[0273] Finally, TCR sequencing analysis revealed particularly significant clonal expansion within brain T cells, with 9.8% and 6.7% of brain T cells showing clonal expansion at 1W and 12W, respectively. This phenomenon was not particularly observed in splenic T cells (Fig. 2l, m). These expanded clones were primarily located within the "proliferative" and "activated" subgroups of the brain (Fig. 2l, m). Clonal expansion was also observed in brain Tconv and CTLs, but this expansion occurred mainly at the later stage (12W) (not shown). Quantitative clonal analysis showed that the clonality of brain resident T cells (Treg) was significantly higher at 1 week of age (1W), which was distinctly different from the clonality observed in lymphoid organs (not shown). This pattern was not observed in other T cell populations (not shown). These unique clonal dynamics highlight the unique selective pressures and mechanisms that promote the specialization of early T cells (Tregs) within the central nervous system, supporting a model in which brain-resident T cells are established early in life and maintained throughout development through specialized adaptive processes. These data demonstrate that brain-resident T cells acquire unique tissue-resident identity, functional specialization, and stable resident status during early postnatal development, suggesting that these cells play a crucial role in brain development and function.

[0274]

[0275] 3. Loss of brain Treg cells impairs central nervous system immune homeostasis and neurodevelopment.

[0276] Considering the important role of Treg cells in maintaining tissue-specific immune niches, we investigated whether neonatal brain Treg cells regulate central nervous system immune homeostasis. To this end, Foxp3 DTRTreg cells were selectively depleted in mice by administering diphtheria toxin (DT) at 8 months of age (P8) and 15 months of age (P15). As a result, the frequency of brain Treg cells was effectively reduced for at least 4 days after treatment (Fig. 3a). Surprisingly, neonatal Treg cell depletion induced a robust accumulation of immune cells within the brain parenchyma, but showed no significant change in the number of immune cells within the dura mater (Fig. 3b). Among the infiltrating cell population, αβ T cells, which are primarily CD62L−CD44+ effector cells, showed a significant increase in both number and proportion, while other lymphocyte subtypes were minimally affected (Fig. 3c). Intravenous anti-CD45 labeling confirmed that these infiltrating T cells were located extravascularly in the parenchyma, and this was further verified by immunohistochemical staining confirming their presence beyond CD31+ vessels (not shown). Consistent with these observations, most of the CD45+ immune cells infiltrating the parenchyma were identified as CD3+ T cells, which are different from the cells located in central nervous system-related compartments such as the choroid plexus or pia mater (Fig. 3d).

[0277] To determine whether the accumulation of T cells in the brain following neonatal Treg reduction is a stochastic or site-specific phenomenon, immunohistochemical analysis was performed on serial sagittal brain sections registered in the Allen Brain Atlas (not shown). In particular, T cell infiltration was highly localized to specific cortical regions, particularly the post-ampullary cortex, rather than being diffusely distributed, and the expression of T cell recruitment chemokines such as CCL11 and CXCL9 was increased (Figs. 3e–g). To further characterize the infiltrating T cells, an ex vivo stimulation analysis comparing brain-infiltrating T cells with spleen T cells was performed (Fig. 3h). Brain-derived T cells showed increased production of Th1 and Th2-related cytokines, specifically IFNγ derived from the CD4+ and CD8+ subgroups and IL-5 derived from the CD4+ subgroup (Figs. 3i, j). These cytokine profiles highlight the activation-effector phenotype of central nervous system infiltrating T cells induced by neonatal Treg reduction. To determine whether local depletion of brain-resident Tregs alone could induce such T cell accumulation, DT was administered directly to the logarithmic tank on the 8th day after birth. Despite the rapid regrowth of Tregs, this partial and local depletion preferentially increased effector T cell accumulation specifically within the brain without affecting peripheral tissues. This further supports the idea that neonatal brain-resident Tregs play a tissue-specific regulatory role in central nervous system immune homeostasis (not shown).

[0278] To evaluate the long-term neuroimmunological effects of neonatal Treg depletion, single-nuclear RNA sequencing (snRNA-seq) was performed on the postampullary cortex, the region with the highest immune infiltration. Neonatal Treg depletion increased the proportion of glial cells compared to the control group (not shown), and astrocytes exhibited inflammatory and immature transcriptional characteristics (not shown). Microglia showed increased expression of "disease-associated microglia" genes, previously known to be associated with increased phagocytosis, as well as genes related to IFNγ and IL-4 signaling responsiveness (not shown). These results suggest sustained neuroimmunological activation following early developmental impairment of brain-resident T cells, highlighting the critical role of T cells in maintaining central nervous system immune homeostasis and glial function.

[0279]

[0280] 4. Neonatal Treg reduction disrupts neuroimmune interactions and impairs spatial memory.

[0281] Considering the snRNA-seq results and the established responsiveness of microglia to immune-derived cytokines, we hypothesized that the activated microglia observed after neonatal Treg reduction may be induced by cytokines secreted by accumulated T cells. Indeed, immunohistochemical analysis confirmed significant microglia activation within the brain parenchyma following neonatal Treg reduction, characterized by morphological changes and increased expression of MHC class I and II molecules (Figs. 4a-d). During critical developmental periods, such abnormal microglia activation is associated with excessive synaptic pruning and consequent neurological dysfunction. Consistent with this, synaptic engulfment by activated microglia was increased, and PSD-95+ postsynaptic puncta were significantly reduced in the cortical regions affected by Treg-reduced mice (Figs. 4e, f).

[0282] To directly confirm whether brain-infiltrating T cells induce microglia activation, an in vitro co-culture analysis was performed comparing primary microglia cultured with brain-derived T cells and spleen-derived T cells (Fig. 4g). Microglia co-cultured with brain-derived T cells showed significantly increased phagocytic activity and higher MHC molecule expression compared to microglia cultured with spleen T cells (Fig. 4h). Consistent with cytokine-mediated mechanisms, phagocytic activity was promoted when microglia were treated with Th1 and Th2 cytokines (IFNγ, IL-4, IL-13), and treatment with IFNγ alone was sufficient to strongly induce MHC expression (not shown). Furthermore, neutralization of IFNγ and IL-4 effectively attenuated T cell-induced microglia activation (Fig. 4h), which comprehensively highlighted cytokine signaling of infiltrating T cells as an important mediator of microglia activation and subsequent abnormal synaptic pruning following neonatal Treg cell depletion.

[0283] After confirming cytokine-induced microglia activation and abnormal synaptic pruning following neonatal Treg cell depletion, the functional effects of these neuroimmunomodulatory disorders on neurons were investigated by electrophysiologically recording neural activity in the posterior ampullary cortex using a Neuropixels probe (Fig. 4i). Treg cell-depleted mice showed a significantly reduced neural firing rate (Fig. 4j) and decreased burst activity essential for synaptic strengthening and spatial encoding (not shown). Furthermore, focal field potential analysis revealed a significant decrease in type 1 theta oscillations (8–12 Hz), a rhythm playing a crucial role in spatial navigation and memory functions within the posterior ampullary cortex circuit (not shown). Complementary snRNA-seq analysis revealed significant transcriptional changes in excitatory neurons rather than inhibitory neurons, and an abundance of synapse-related pathways such as "synaptic organization," which is consistent with the compensatory mechanism induced by excessive synaptic pruning (not shown). In particular, genes associated with neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease, were significantly upregulated in T cell-depleted brains, which suggests long-term neuronal vulnerability following immune dysregulation in early infancy (not shown).

[0284] With the role of the posterior protuberance in spatial cognition established, the cognitive and behavioral consequences of neonatal T cell depletion were further evaluated. Mice with depleted Treg cells exhibited normal anxiety-like behaviors, social interactions, and stress responses (not shown), but showed selective deficits in spatial memory tasks, which was evidenced by impaired recognition of new locations but no issues with the recognition of new objects (Fig. 4k). Additionally, in a Y-maze voluntary alternation experiment, mice with depleted Treg cells showed a significant decrease in spatial working memory, characterized by an increased frequency of revisiting previously explored arms (Fig. 4k). Collectively, these data demonstrate that depletion of resident Treg cells in the neonatal brain induces sustained microglia activation and abnormal synaptic remodeling by T cells, resulting in neural circuit damage and selective spatial memory deficits. These results indicate that resident Treg cells in the neonatal brain play an essential role in maintaining appropriate neuroimmunological interactions necessary for neural circuit formation and cognitive integrity.

[0285]

[0286] 5. Increase in brain-specific Tregs alleviates neuroinflammation and improves neurodevelopmental disorder behaviors.

[0287] Following neonatal Treg deficiency, dysregulation of synaptic pruning and abnormal activation of microglia—major neuroimmunological changes widely associated with neurodevelopmental disorders, including autism spectrum disorder (ASD)—were prominently observed. Therefore, the present invention hypothesized that selectively increasing brain-resident Tregs could restore central nervous system immune homeostasis and improve behavioral deficits associated with neurodevelopmental disorders. To verify this hypothesis, we used BTBR mice, a validated ASD model characterized by significant neuroinflammation and behavioral disorders (not shown). We selectively amplified brain Tregs by delivering an IL-2 gene targeting astrocytes using the AAV-PHP.eB vector under the GFAP promoter (AAV-GFAP-mIL2). This gene was administered at 5 weeks of age, a therapeutically critical developmental period similar to human childhood and adolescence (Fig. 5a).

[0288] The increase in brain-specific Tregs significantly increased the expression frequency of central nervous system Tregs without significantly affecting the expression frequency of peripheral or meningeal Tregs, confirming that IL-2 delivery targeting astrocytes is effectively targeted to the central nervous system (Fig. 5b). Consequently, neuroinflammation in BTBR mice was significantly reduced, which manifested as a decrease in the expression frequency of activated IFNγ-producing CD8+ T cells (not shown). Behavioral tests revealed that treated BTBR mice showed significant improvements across core ASD-like behaviors, as well as reductions in anxiety and compulsive behaviors and improved social interactions (Figs. 5c–e). In particular, behavioral improvement was closely correlated with an increase in the frequency of brain-resident Tregs, which further emphasizes that brain Tregs play a crucial role in regulating neuroimmune interactions and improving behavioral abnormalities (Fig. 5f). Importantly, these therapeutic effects were sustained for a long period, as evidenced by continued behavioral improvement and a stable increase in brain-resident Tregs even at the third month after treatment, which highlights the sustained benefits of brain-targeted Tregs (not shown).

[0289] Considering the established association between immune dysfunction and neuroinflammation in ASD, the peripheral immune profiles of children diagnosed with ASD and normal developmental controls (TDC) were further evaluated. Although the total Treg frequencies were similar between the two groups, ASD patients had a higher proportion of inactive Tregs and an increased frequency of cytotoxic CD8+ T cells producing IFN-γ and Granzyme B (GZMB) (Figs. 5g–i). Importantly, the proportion of inactive Treg cells showed a positive correlation with ASD severity (SRS score), whereas the frequency of activated CD8+ T cells showed a specific correlation with the mannerism subscale score, highlighting a significant immune-behavioral association in ASD (Fig. 5j).

[0290] To further evaluate the potential for Treg modulation, a preliminary clinical trial using low-dose IL-2 (LdIL-2) therapy was conducted in children with ASD (Fig. 5k). LdIL-2 treatment significantly increased the proportion of peripheral Treg cells, decreased the Tc1 / Treg cell ratio, and reduced inflammatory cytokines including IL-17F and IL-22, indicating the restoration of peripheral immune balance (Fig. 5l). Clinically, children treated with LdIL-2 demonstrated significant behavioral improvement, which was manifested by reductions in scores on the Child Autism Assessment Scale (CARS), the Autism Treatment Assessment Checklist (ATEC), and the Abnormal Behavior Checklist (ABC). Behavioral improvement persisted during the 3-month follow-up, and the increase in peripheral Treg cells, along with these behavioral improvements, continued even after repeated administration of LdIL-2 (Fig. 5m). Importantly, an increase in peripheral Treg cells showed a negative correlation with behavioral symptom scores, which strongly supports the Treg-mediated immune regulatory mechanisms underlying the clinical improvement of ASD (Fig. 5n). These experimental results demonstrate that an increase in brain-specific Treg cells effectively alleviates neuroinflammation, restores central nervous system immune homeostasis, and improves behavioral deficits characteristic of neurodevelopmental disorders. Furthermore, these findings highlight that brain-resident T cells are central regulators of neuroimmune interactions and emphasize their potential as sustainable therapeutic targets for immune-mediated neurodevelopmental disorders.

[0291]

[0292] [conclusion]

[0293] This invention revealed that brain-resident regulatory T cells are important regulators of central nervous system immune homeostasis during early neurodevelopment. Brain T cell depletion in newborns resulted in the abnormal accumulation of activated αβ T cells, which appeared preferentially in specific cortical regions, particularly the post-ampullary cortex. This localized immune dysregulation caused pathological microglia activation, increased MHC expression, and excessive synaptic pruning, ultimately leading to persistent impairment of neural circuits and spatial cognition. Conversely, selective astrocyte-targeted IL-2 amplification of brain-resident regulatory T cells restored central nervous system immune homeostasis and significantly improved core behavioral disorders in an ASD BTBR mouse model. Importantly, low-dose IL-2 treatment in human ASD patients enhanced peripheral regulatory T cell function, reduced inflammatory cytokines, and significantly improved behavioral symptoms, presenting the first clinical evidence that targeting regulatory T cells can improve neuroimmunological dysfunction and behavioral symptoms in ASD. A comprehensive review of these experimental results confirms that neonatal brain-resident T cells are central regulators of central nervous system immune privilege and highlights their therapeutic potential in immune-mediated neurodevelopmental disorders.

[0294] Previous studies emphasized the dominance of innate immune cells in the neonatal meninges and suggested that early adaptive immunity is limited within the central nervous system barrier compartment; however, the results of this study confirmed that the deep regions of the central nervous system parenchyma constitute the early adaptive immune niche. The unique transcriptional profiles and tissue-specific clonal expansion observed in brain-resident T cells strongly suggest local antigen-driven mechanisms supporting early central nervous system colonization. Consistent with this, recent studies have identified central nervous system-derived endogenous peptides presented via MHC-II molecules as essential regulators of central nervous system-specific immune cell localization and T cell development, highlighting that antigen specificity is a key determinant of central nervous system immune privilege and tissue-resident cell identity. Consequently, the precise molecular identity of the central nervous system-derived antigens that induce brain T cell specificity has not yet been elucidated.

[0295] The results of this experiment demonstrate that T cells infiltrating after neonatal Treg deficiency interact with microglia to induce sustained microglia activation and excessive synaptic pruning, suggesting important adaptive immune regulation regarding neuroimmunological interactions during early central nervous system development. Recent studies have shown that adaptive immune cells, particularly CD4+ T cells, directly regulate microglia-mediated synaptic microneedling and neural circuit formation. Similarly, brain-resident Tregs can exert direct regulatory effects on central nervous system resident cells, a finding supported by ligand-receptor interaction analysis (not shown), which identified several candidate signaling pathways mediating these interactions. These include semaphorin-flexin interactions (e.g., Sema4d-Plxnb1 / 2), which are known to influence axon induction, integrin signaling, and synaptic plasticity in the developing nervous system. VCAM1-integrin binding (e.g., VCAM1-Itga4 / Itgb1 / Itgb7) mediates immune cell adhesion and transport across the BBB, while laminin-integrin interactions (Lamc1-Itgav / Itgb8) regulate cell adhesion and microglia migration. Furthermore, studies using antigen-specific TCR transfection models have revealed that sustained antigen-specific interactions between microglia MHC-II and regulatory T cell (Treg) TCRs are essential for the differentiation of brain-resident regulatory Treg cells and stable central nervous system residence, highlighting the antigen-dependent mechanisms underlying the mechanisms of neuroimmunological regulation.

[0296] Clinically, the results of this study have significantly advanced treatment strategies for Autism Spectrum Disorder (ASD) by being the first to demonstrate therapeutic efficacy targeting regulatory Treg cells in both preclinical and clinical settings. By utilizing astrocyte-targeted IL-2 gene delivery (AAV-GFAP-mIL-2), the sustained proliferation of central nervous system resident Tregs was selectively achieved in a BTBR mouse model, effectively reducing neuroinflammation and improving key ASD-related behaviors. Interestingly, preclinical data showed that peripheral administration of an IL-2 / anti-IL-2 complex significantly increased both peripheral and brain-resident Treg populations, suggesting the potential for low-dose IL-2 treatment to similarly proliferate brain-resident Tregs clinically in humans (not shown). Supporting this hypothesis, this preliminary clinical study applied these results to human ASD patients and demonstrated that low-dose IL-2 treatment successfully enhanced Treg number and function, reduced inflammatory cytokines, and significantly improved behavioral symptoms. This is the first clinical evidence demonstrating the efficacy of immune-based therapy through Treg modulation in ASD, and shows that immune dysregulation is a treatable target for neurodevelopmental disorders. Utilizing engineered IL-2 mutane, which has demonstrated enhanced Treg specificity and safety in recent clinical studies, will further enable sustained, specific, and potent proliferation of brain-resident Tregs in human patients.

[0297]

[0298] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for the treatment or prevention of developmental disorders, comprising interleukin (IL) or a pharmaceutically acceptable salt thereof as an active ingredient.

2. In Paragraph 1, A pharmaceutical composition for the treatment or prevention of developmental disorders, wherein the above interleukin is one or more selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, and IL-25.

3. In Paragraph 2, A pharmaceutical composition for the treatment or prevention of developmental disorders, wherein the above interleukin is one or more selected from the group consisting of IL-2, IL-2 mutain, TGF-beta, and IL-10, which activate the function of regulatory T cells.

4. In Paragraph 3, A pharmaceutical composition for the treatment or prevention of developmental disorders, wherein the above interleukin is low-dose IL-2 (LdIL-2).

5. In Paragraph 4, A pharmaceutical composition for the treatment or prevention of developmental disorders, wherein the low dose IL-2 is IL-2 at a dose of 10,000 to 25,000 IU / kg.

6. In Paragraph 5, A pharmaceutical composition for the treatment or prevention of developmental disorders, wherein the above low dose IL-2 is injected every other day for two weeks.

7. In Paragraph 6, A pharmaceutical composition for the prevention or treatment of developmental disorders, wherein the above developmental disorder is a disorder selected from the group consisting of intellectual disability, cerebral palsy, pervasive developmental disorder (PDD), developmental language disorder, dysfunction of special senses including sight or hearing, learning disability, attention deficit hyperactivity disorder, epilepsy, and combinations thereof.

8. In Paragraph 7, A pharmaceutical composition for the prevention or treatment of developmental disorders, wherein the above-mentioned pervasive developmental disorder is a disorder selected from the group consisting of autism spectrum disorder (ASD), Asperger syndrome, childhood disintegrative disorder, Rett's syndrome, atypical autism spectrum disorder, and combinations thereof.

9. Adenovirus recombinant vector containing an interleukin gene.

10. In Paragraph 9, The adenovirus recombinant vector, wherein the above interleukin is one or more selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, and IL-25.

11. In Paragraph 10, The above-mentioned interleukin is one or more selected from the group consisting of IL-2, IL-2 mutain, TGF-beta, and IL-10, which activate the function of regulatory T cells, in an adenovirus recombinant vector.

12. An adenovirus cell line transfected with the adenovirus recombinant vector of any one of claims 9 to 10.

13. In Paragraph 12, The above adenovirus is an adenovirus cell line that is an adeno-associated virus (AAV).

14. A pharmaceutical composition for the prevention or treatment of developmental disorders comprising an adenovirus recombinant vector of any one of claims 9 to 10 as an active ingredient.

15. In Paragraph 14, A pharmaceutical composition for the prevention or treatment of developmental disorders, wherein the above developmental disorder is a disorder selected from the group consisting of intellectual disability, cerebral palsy, pervasive developmental disorder (PDD), developmental language disorder, dysfunction of special senses including sight or hearing, learning disability, attention deficit hyperactivity disorder, epilepsy, and combinations thereof.

16. In Paragraph 15, A pharmaceutical composition for the prevention or treatment of developmental disorders, wherein the above-mentioned pervasive developmental disorder is a disorder selected from the group consisting of autism spectrum disorder (ASD), Asperger syndrome, childhood disintegrative disorder, Rett's syndrome, atypical autism spectrum disorder, and combinations thereof.

17. A method for treating or preventing a developmental disorder, comprising the step of administering interleukin (IL) or a pharmaceutically acceptable salt thereof to a patient with a developmental disorder.

18. In Paragraph 17, A method for treating or preventing developmental disorders, wherein the above interleukin is one or more selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, and IL-25.

19. In Paragraph 18, A method for treating or preventing developmental disorders, wherein the above interleukin is one or more selected from the group consisting of IL-2, IL-2 Mutain, TGF-beta, and IL-10, which activate the function of regulatory T cells.

20. In Paragraph 3, A method for treating or preventing developmental disorders, wherein the above interleukin is low-dose IL-2 (LdIL-2).

21. In Paragraph 20, A method for treating or preventing developmental disorders, wherein the above low-dose IL-2 is IL-2 at a dose of 10,000 to 25,000 IU / kg.

22. In Paragraph 21, A pharmaceutical composition for the treatment or prevention of developmental disorders, wherein the above low dose IL-2 is injected every other day for two weeks.

23. In Paragraph 22, A method for treating or preventing a developmental disorder, wherein the above developmental disorder is a disorder selected from the group consisting of intellectual disability, cerebral palsy, pervasive developmental disorder (PDD), developmental language disorder, dysfunction of special senses including sight or hearing, learning disability, attention deficit hyperactivity disorder, epilepsy, and combinations thereof.

24. In Paragraph 23, A method for treating or preventing a developmental disorder in which the above-mentioned pervasive developmental disorder is a disorder selected from the group consisting of autism spectrum disorder (ASD), Asperger syndrome, childhood disintegrative disorder, Rett's syndrome, atypical autism spectrum disorder, and combinations thereof.

25. A method for preventing or treating a developmental disorder, comprising the step of administering an adenovirus recombinant vector containing the IL-2 gene to a patient with a developmental disorder.

26. In Paragraph 25, A method for preventing or treating a developmental disorder, wherein the above developmental disorder is a disorder selected from the group consisting of intellectual disability, cerebral palsy, pervasive developmental disorder (PDD), developmental language disorder, dysfunction of special senses including sight or hearing, learning disability, attention deficit hyperactivity disorder, epilepsy, and combinations thereof.

27. In Paragraph 26, A method for preventing or treating a developmental disorder in which the above-mentioned pervasive developmental disorder is a disorder selected from the group consisting of autism spectrum disorder (ASD), Asperger syndrome, childhood disintegrative disorder, Rett's syndrome, atypical autism spectrum disorder, and combinations thereof.

28. A method for promoting astrocyte-targeted IL-2 expression based on a CNS resident cell specific (GFAP, Synapsin, CamKII) promoter.

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