Pharmaceutical compositions for the identification and treatment of pathological neurodegeneration and age-related cognitive decline.

Abstract

To provide pharmaceutical compositions for the diagnosis, prevention, and / or treatment of age-related cognitive decline, including pathological neurodegeneration. [Solution] A pharmaceutical composition comprising the following components: a therapeutically effective amount of one or more of the following: a CD103 inhibitor, a perforin 1 inhibitor, and an IFNγ inhibitor, wherein the CD103 inhibitor is an anti-CD103 antibody, selected from the group consisting of PE anti-human CD103 antibody derived from clone Ber-ACT8, mouse anti-human CD103 monoclonal antibody (mAb) derived from clone 2G5.1, humanized antibody of 2G5.1, OX-62, humanized antibody of OX-62, anti-mouse CD103 mAb derived from clone 2E7, humanized antibody of 2E7, and paxilin; the perforin 1 inhibitor is selected from the group consisting of diarylthiophene and GSK2126458; and the IFNγ inhibitor is selected from the group consisting of mesopram and locagramide.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under § 119(e) of the United States Patent Act to U.S. Provisional Patent Application No. 62 / 630129, filed on 13 February 2018, and the entire contents of that Provisional Patent Application are incorporated herein by reference.

[0002] Field of Invention This invention relates to the diagnosis, prevention, and treatment of age-related neurodegeneration or pathological neurodegeneration. [Background technology]

[0003] Aging is a contributing factor to the onset and / or progression of several diseases, but the specific ways in which aging affects the dynamics of individual diseases remain largely a mystery. Chronic inflammation is increasingly recognized as a significant contributing factor to age-related diseases, which include cancer, cardiovascular diseases, and neurological conditions such as stroke, trauma, and neurodegeneration.

[0004] T cells are the primary regulators of inflammation throughout the body, and their misregulation contributes to chronic inflammation. CD8+ T cells can acquire self-destructive capabilities, particularly when the peripheral T cell pool is depleted and these cells undergo homeostatic proliferation. Such depletion occurs gradually with aging due to thymic regression while producing new T cells, or more rapidly due to stress, trauma, or infection. Age-related T cell proliferation persists into late middle age in humans and can occur anywhere, making it difficult to study whether pathological factors, rather than natural aging processes, are involved in abnormal T cell proliferation. Furthermore, abnormal age-related T cell proliferation is relatively rare in rodent laboratory animals, and even when it does occur, its functional effects are often offset by sustained thymic activity in aging rodents.

[0005] When T cells are introduced into hosts with reduced lymphocyte counts, homeostatic proliferation occurs rapidly in those hosts, and in rodent experimental animals, the resulting cells can sometimes enhance artificial autoimmunity. Nevertheless, the relationship between this more rapid proliferation and age-related T cell abnormalities is not very clear, and in most cases, it is not known that recognizable age-related diseases are exacerbated by this relationship.

[0006] Abnormal CD8+ T cell clones proliferate, particularly in most aging humans, while this process is counteracted by compensatory processes in aging mice. Furthermore, changes in memory CD8+ T cells are among the clear physiological differences observed between mouse models and human Alzheimer's disease, with these cells decreasing in mouse models and increasing in human Alzheimer's disease. Recent studies have also convincingly demonstrated an increase in memory CD8+ T cells in the circulatory and / or central nervous system (CNS) of Alzheimer's patients. These increases are commonly accompanied by significant changes in other T cells, and tend to correlate more strongly with tauopathy and / or cognitive decline. Therefore, age-related homeostatic proliferation and the resulting abnormal memory CD8+ T cells may be unknown physiological factors in mice that could influence resistance to the overall pathogenesis of Alzheimer's disease. However, investigating this remains difficult due to the widespread presence of abnormally aging CD8+ T cells in healthy humans, their scarcity in mice, and the fact that these cells arise in all species along with other aging characteristics.

[0007] Therefore, one object of the present invention is to provide a composition for the diagnosis, prevention, and / or treatment of age-related cognitive decline, including pathological neurodegeneration, and a diagnostic method, preventive method, and / or therapeutic method using the composition.

[0008] Another object of the present invention is to provide compositions for screening candidate therapeutic agents, preventive agents, and / or diagnostic agents for human cognitive decline in an in vitro system or rodent animal model, and a screening method using the same composition.

[0009] All publications within this specification are referred to by reference to the same extent as individual publications or patent applications are specifically and individually indicated as being referred to by reference. The following descriptions contain information that may be useful in understanding the present invention. This does not constitute an admission that any of the information provided herein is prior art or relating to the invention claimed in this application, nor does it constitute an admission that any of the publications specifically or expressly referenced are prior art. [Overview of the Initiative]

[0010] The following embodiments and their aspects are described and illustrated together with the compositions and methods, but they are representative and illustrative and not intended to limit the scope of the invention.

[0011] To protect against cognitive decline and / or reduce the severity of cognitive decline in elderly subjects at risk, subjects with mild cognitive impairment, and / or subjects with pathological neurodegeneration, CD103 inhibitors, CD8+T RM The present invention provides a method comprising administering an effector molecule inhibitor of (CD8+ commensal memory T cells) and / or an immunotolerogenic vaccine. In various embodiments, CD8+ T RM CD8+T can be reactive or specific to amyloid precursor protein (APP) or APP peptide. According to one embodiment of this method, by administering a vaccine containing a CD103 inhibitor, a perforin 1 inhibitor, an interferon-gamma (IFNγ) inhibitor, and / or APP peptide, CD8+T can be reactive to APP or APP peptide compared to before administration of one or more of the above inhibitors or one of the above vaccines, or compared to a control subject who has not been administered one or more of the above inhibitors. RM It is possible to suppress the binding or reaction of these molecules.

[0012] These inhibitors include antibodies or antibody fragments, small molecules, or nucleic acids. In some embodiments, the inhibitor can be an anti-CD103 antibody such as 2G5.1, a mouse anti-human IgG2a monoclonal antibody, or a humanized antibody of 2G5.1. In other embodiments, the inhibitor can inhibit perforin 1 or interferon γ. In some embodiments, the tolerogenic vaccine can include an amyloid precursor protein or a peptide thereof.

[0013] A method for identifying a subject who is prone to, or has, age-related neurodegeneration including Alzheimer's disease, the method including detecting an elevated level of CD103+ resident memory T cells (T RM ) in the blood is provided.

[0014] Before, during, and / or between treatments for memory impairment, a sample collection device for collecting and quantifying CD103+ CD8+ T RM cells from a subject, and optionally an operation manual, are provided in a kit.

[0015] A system for identifying and / or screening candidate therapeutic, prophylactic, and / or diagnostic agents for human cognitive decline or age-related neurodegeneration, the system including a CD44 hi CD123 + CD127 hi KLRG1 + CD103 + resident memory CD8+ T cell phenotype obtainable from a rodent (e.g., a mouse) is provided. In some embodiments, this CD44 hi CD123 + CD127 hi KLRG1 + CD103 + resident memory CD8+ T cell phenotype can be obtained by administering resident memory CD8+ T cells to thymus-deficient mice.

[0016] A method for identifying and / or screening candidate drugs for the treatment or prevention of age-related neurodegeneration in humans is to test the candidate drug in vitro using the above CD44 hi CD123 + CD127 hi KLRG1 + CD103 + By contacting resident memory CD8+ T cells, or the above CD44 hi CD123 + CD127 hi KLRG1 + CD103 + This may include administering the candidate drug to an animal model containing resident memory CD8+ T cells and identifying the level of reduction in CD103-positive resident memory CD8+ T cells, the level of reduction in effector molecules of those cells, or the reduction in the amount of CD8+ T cell migration from the peripheral system to the brain of the animal.

[0017] Further features and advantages of the present invention will become apparent from the following detailed description, along with the accompanying drawings illustrating various aspects of embodiments of the present invention.

[0018] The reference drawings illustrate exemplary embodiments. The embodiments and drawings disclosed herein should be considered illustrative rather than limiting. [Brief explanation of the drawing]

[0019] [Figure 1A]Figures 1A to 1G show that hiT cells exhibit the age-related commensal memory phenotype (hiTRM). Figures 1A to 1D show results from spleen specimens, and Figures 1E to 1G show results from brain analysis. Figure 1A shows representative flow cytometry analysis of aging markers in spleen CD8+ T cells derived from young (less than 10 weeks) and elderly (over 12 months) C57BL / 6 (B6) mice (labeled "Young B6" and "Elderly B6," respectively), and young (6 weeks) B6.Foxn1 recipients 3 to 5 weeks after intravenous infusion of CD8+ T cells (labeled "CD8→B6.Foxn1"). Figure 1B shows the percentage of lymphocytes derived from flow cytometry collected from 6 or more mice per group, and the mean fluorescence intensity (Figures 1C and 1D). The proportion of B6 mice possessing a specific D→J segment in the brains of young (<10 weeks, labeled "Young B6"), middle-aged (6 months, labeled "Middle-aged B6"), and elderly (over 12 months, labeled "Elderly B6") mice, exhibiting "diverse" TCRVβ chain D→J gene segment utilization (more than 3 segments per brain), shows an age-dependent pattern of progressive decrease in diversity and increase in specific D→J segment utilization (i.e., clonality; Figures 1E and 1F; columns appear in the order defined from left to right at the bottom of each figure). D→J diversity and segment utilization were significantly correlated only between the brains of elderly B6 mice and the brains of young CD8→B6.Foxn1 mice (Figure 1G). *P<0.05, **P<0.01, ***P<0.005. Flow cytometry markers were analyzed using two-sided t-tests with more than 5 mice per group in three or more independent studies. For PCR compilation, Pearson's correlation was used with 10 or more mice per group. [Figure 1B] Same as above [Figure 1C] Same as above [Figure 1D] Same as above [Figure 1E] Same as above [Figure 1F] Same as above [Figure 1G] Same as above [Figure 1H]Figures 1H–1K show the proliferation of donor cells in amyloid precursor protein (APP)-deficient B6.Foxn1 mice. Purified CD8+ T cells derived from female C57BL / 6 or genetic knockout hosts were injected into 8–10-week-old female B6.Foxn1 recipients, B6.Foxn1-AppKO recipients, or B6.CD45.1 genetic recipients (Figure 1H). Blood was analyzed by flow cytometry after 3 days using the gate setting shown (Figure 1I) and antibodies against T cell markers, and the percentage of CD3ε+CD8+ cells in the gated cells (%) was summarized in (Figure 1J). B6.Foxn1 mice were crossed with B6.App-knockout mice, and homozygous double mutants (B6.Foxn1-AppKO) were identified by PCR and phenotypic analysis at Jackson Laboratories (Bar Harbor, Maine). CD8+ T cell proliferation was then evaluated in B6.Foxn1 female recipients and B6.Foxn1-AppKO female recipients by CFSE dilution (Figure 1K; n=3 B6.Foxn1 mice and n=5 B6.Foxn1-AppKO mice; *P<0.04, ***P<0.00001 by two-sided t-tests in three independent trials; n≧5 mice / group in three or more independent trials for all markers). [Figure 1I] Same as above [Figure 1J] Same as above [Figure 1K] Same as above [Figure 2A]Figures 2A to 2E show how hiTRM reacts to autoantigens and selectively invades the brain (i.e., brain CD8+ T cell phenotype after transfer to nude mice). Figure 2A shows the light scatter and gate setting of brain lymphocytes and CD8+ T cells in B6.Foxn1 recipients. Figures 2B and 2C show the percentage and phenotype of CFSE+CD8+ T cells in brain lymphocytes in B6.Foxn1 recipients 3 days (Figure 2B) and 10 weeks (Figure 2C) after injection. Figures show enhancement of staining with a pMHC I multimer (a custom dextramer synthesized by Immudex USA in Fairfax, Virginia) for Trp-2-DCT(180-188) / H-2Kb epitopes and APP(470-478) / H-2Db epitopes on KLRG1+CD8+ T cells in the brain (Figures 2D and 2E) and spleen (Figure 2E) of B6.Foxn1 recipients 10 weeks after injection (*P<0.05 by two-sided t-tests in three or more independent studies; n>6 for all analyses, significant compared to the PBS group). [Figure 2B] Same as above [Figure 2C] Same as above [Figure 2D] Same as above [Figure 2E] Same as above [Figure 2F]Figures 2F-2J show that hiTRM induction increases CD8 and amyloid precursor protein (APP) / Ab in the brain (i.e., PCR and Western blotting of T cell markers and amyloid markers). PCR of the D1→J1 gene segment (Figure 2F) and D2→J2 gene segment (Figure 2G) of the TCRVβ chain showed a diverse T cell repertoire in young and aged C57BL / 6 (B6) mice, but limited TCRVβ chain diversity was observed in B6.Foxn1 recipients of CD8+ T cells (CD8→B6.Foxn1) after 10 weeks. In B6.Foxn1 mice, if wild-type CD8+ T cells had not been previously injected, there were no TCR products reconstituted in the brain (i.e., visually confirmed to contain only germline "G") (Figures 2F and 2G; note: segment J2.6 is a pseudogene). Figure 2 shows Western blots of CD8α (antibody clone 2.43; Figure 2H) and β-amyloid (antibody clone 4G8; Figure 2I) in the hippocampus of excised brain tissue from B6.Foxn1 hosts that had undergone 10 weeks of adoptive transfer of CD8+ T cells derived from young (less than 5 months) C57BL6 (B6) and young (6-8 weeks) B6 donors, and B6.Foxn1 hosts that had not undergone adoptive transfer. CD8 protein is detectable at very low levels in B6, but is undetectable in B6.Foxn1 unless wild-type CD8=T cells were injected 10 weeks prior. "Ref" = Spleen DNA or cell lysates from 6-10 week old female C57BL / 6 used in the analysis. Figure 2J shows the timeline of the experiment. [Figure 2G] Same as above [Figure 2H] Same as above [Figure 2I] Same as above [Figure 2J] Same as above [Figure 3A]Figures 3A–3J show Aβ plaques and neurofibrillary disease symptoms in nude mice with hiT cells. Figure 3A shows Western blots of surfactant-soluble APP cleavage products (APPCl) in excised cortical and hippocampal cells 3 weeks after control or cell injection (→) in the indicated recipients. The cells / control recipients in Figures 3B–3J are exclusively B6.Foxn1, and unless otherwise indicated, they are 15 months after injection. Figure 3B shows forebrain ELISA of the Triton-soluble fraction Aβ1-40 / 42. Figure 3C shows parenchymal plaques with and without p-tau or curcumin counterstaining in the indicated mouse groups, and Figure 3D summarizes the 4G8 area percentages in the entorhinal (Ent) cortex, cingulate (Cng) cortex, and hippocampus (Hippo). Figure 3E shows forebrain Western blots of surfactant-soluble phosphotau (p-tau) and paired helical fibrils (PHF), and a summary of signal quantification (Figure 3F). Figure 3G shows the brain and silver-stained cells in 18-month-old ADtg (Tg2576) mice, including an inset of serial p-tau → Gallius staining. Figure 3H summarizes the percentage of Gallius+ neurons, a figure showing astrocytes (Gfap+), and a figure showing microglia (Iba-1+; Figures 3I and 3J). Two-sided t-tests in three or more independent studies for all analyses showed *P<0.05, **P<0.01, and ***P<0.005 compared to the PBS group. [Figure 3B] Same as above [Figure 3C] Same as above [Figure 3D] Same as above [Figure 3E] Same as above [Figure 3F] Same as above [Figure 3G] Same as above [Figure 3H] Same as above [Figure 3I] Same as above [Figure 3J] Same as above [Figure 3K]Figures 3K and 3L illustrate the induction of fibrillary inclusions within brain cells by hiTRM at 6 months of age (i.e., separate staining for curcumin and thioflavin S in the dentate gyrus of nude mice with hiT cells). Hippocampal sections from the indicated groups (all B6.Foxn1 recipients except for AD-Tg=Tg2576 mice) were stained with 4G8(Aβ) and curcumin 6 months after intravenous control / cell injection, or at 14 months of age for AD-Tg mice (Figure 3K). Right-pointing arrows highlight Aβ deposits that are not co-stained with curcumin. Upward-pointing arrows show co-localized Aβ and curcumin deposits, indicating mature senile plaques. Downward-pointing arrows highlight curcumin+ structures that are not co-stained with Aβ, i.e., non-amyloid fibrillary deposits. DAPI was not used in these stains. The background in the blue channel is presented for anatomical reasons only. Figure 3L shows the tracking thioflavin S staining of the dentate gyrus of B6.Foxn1 hiT recipients in the PBS and wild-type CD8 groups 6 months after control / cell injection, and in 20-month-old AD-transgenic (Tg) rats. The brains of AD-Tg rats clearly contain tau PHF (Cohen et al., 2013), so we used this, but our technique did not allow us to stain them with thioflavin S. [Figure 3L] Same as above [Figure 3M] Figures 3M and 3N show silver-stained neural structures in the experimental groups. They show Gallius silver staining in cortical and hippocampal brain regions, illustrating typical neurofibrillary fibrous tissue (NFT) morphology (inset) in wild-type CD8 and IFNγKO-CD8 mice. Background silver staining was occasionally observed in PrfKO-CD8 or PBS mice, but similar NFT morphology was not shown (inset). Individual images are from different mice in each group (Figure 3M). Figure 3N compares Gallius+ structures in the hippocampus (left) and cortex (ctx, right) of a nude mouse (wild-type CD8) with hiT cells to those in the cortex of a human with severe AD (Brack stage VI). All images, magnifications, and scales in Figures 3M and 3N are identical (20x), and the insets are also identical. [Figure 3N] Same as above [Figure 3O] Figures 3O to 3S show brain CD8+ T cells in hiTRM recipient B6.Foxn1 mice. Brain tissue from the hippocampus and cortex of B6.Foxn1 recipients 15 months after injection of wild-type, IFNγKO, or PrfKO CD8+ T cells, or PBS, was co-stained for CD8 and p-tau (inset) (Figure 3O) and quantified. CD8+ cells were mostly isolated, but occasionally interacted with p-tau+ neurons, as seen in Figure 3O (inset). Group data are summarized in Figure 3P. In Figures 3A-3J or 3O-3S, the areas of astrocytes (GFAP, Figure 3Q), microglia (Iba-1, Figure 3R), or CD8+ T cells (CD8, Figure 3S) that changed significantly (**P<0.01, *P<0.05; two-sided t-test compared to PBS control) correlated with the 4G8+ plaque area ratio in each group. The p-values ​​of linear regression and Pearson's correlation (r) are shown (Figures 3Q-3S). [Figure 3P] Same as above [Figure 3Q] Same as above [Figure 3R] Same as above [Figure 3S] Same as above [Figure 4A]Figures 4A–4N show neurodegeneration assessment indices and cognitive function in nude mice with hiT cells (i.e., hiTRM recipient B6.Foxn1 mice). The cell / control recipient in all panels is exclusively B6.Foxn1. Figures 4A and 4B show staining for NeuN and GFAP 15 months after cell / control injection, and Figure 4C shows cell count in the hippocampus. Figures 4D show brain atrophy over time in the PBS and wild-type CD8 groups (amounts normalized to PBS control at each time point). Figure 4E shows a representative forebrain Western blotting, and Figure 4F shows Western blotting signals of NeuN, drebrin, and synaptophycin normalized to GAPDH. Figure 4G shows the correlation of NeuN with brain volume. Figure 4H shows a representative open-field test at 13 months. Figure 4I shows the performance of the fear conditioning test over time, and Figure 4J shows spontaneous alternation behavior (SA) at 12 months. Figures 4K (two-tailed ANOVA P), Figure 4L, and Figures 4M and 4N show Burns maze learning at 14 months (black symbols = P compared to PBS and wild-type CD8, respectively). Two-tailed t-tests are ***P<0.005, **P<0.01, *P<0.05, +P<0.1 unless other tests are indicated. [Figure 4B] Same as above [Figure 4C] Same as above [Figure 4D] Same as above [Figure 4E] Same as above [Figure 4F] Same as above [Figure 4G] Same as above [Figure 4H] Same as above [Figure 4I] Same as above [Figure 4J] Same as above [Figure 4K] Same as above [Figure 4L] Same as above [Figure 4M] Same as above [Figure 4N] Same as above [Figure 5A]Figures 5A-5D illustrate that CD103 deficiency primarily affects CD8+ T cells and brain localization. The figures show flow cytometry (Figure 5A), Western blot (Figure 5B), a summary of Western signaling (Figure 5C), and open-field test results (Figure 5D; columns appear from top to bottom on the right side of this figure) from CD103-deficient (B6.CD103KO) mice and age-matched wild-type (B6) mice (n=8). CD103 deficiency primarily affects CD8+ T cells (Figure 5A), particularly reducing CD8 signaling in the brain (Figures 5B and 5C; CD8+TRM increases particularly in the aging brain when deficiency is absent), and slightly slowing motor function with age (Figure 5D). [Figure 5B] Same as above [Figure 5C] Same as above [Figure 5D] Same as above [Figure 5E] Figures 5E-5H illustrate how CD103 deficiency protects against age-related cognitive decline. They show the Burns maze performance of young and aged CD103-deficient mice and wild-type mice, specifically the training latency (Figure 5E), memory retention latency (Figure 5F), reverse learning latency (Figure 5G), and entry errors during each period (Figure 5H). In this study, the primary age-related impairment in this strain was reported to be entry errors. [Figure 5F] Same as above [Figure 5G] Same as above [Figure 5H] Same as above [Figure 6A]Figures 6A to 6F show elevated hiT cell-related evaluation indices in the brains of human Alzheimer's disease patients. Figure 6A shows GFAP expression units in the brains of non-Alzheimer's disease subjects (AD-free) compared to those in the brains of AD subjects. Figure 6B shows the percentage change in gene expression levels compared to the corresponding GFAP expression levels in the brains of the aforementioned subjects. Figure 6C shows PRF1 Western blotting and immunofluorescence, and Figure 6D shows quantitative analysis in the brains of age-matched normal (n=6), mild (n=5), or severe (n=12) Alzheimer's disease patients. Figure 6E shows Alzheimer's disease brains co-stained with anti-CD8 (Serotec) and APP (471-479) / HLA-A2 multimer (Immudex USA, Fairfax, Virginia), and Figure 6F shows quantitative analysis of epitope-responsive T cells (P=0.002, two-sided t-test). The overall level of CD8+ T cells remained unchanged (1.63±0.29 vs. 2.29±0.55 cells / vascular space in Alzheimer's disease versus normal-aged controls; P=0.31, two-sided t-test). [Figure 6B] Same as above [Figure 6C] Same as above [Figure 6D] Same as above [Figure 6E] Same as above [Figure 6F] Same as above [Figure 7A] Figures 7A and 7B show abnormalities in APP-specific CD8+ TRM levels in patients at risk of age-related or disease-related cognitive decline (MoCA=26-30) and patients with age-related or disease-related cognitive decline (MoCA<26) within all patients (Figure 7A) and specifically within HLA-A2+ patients (potentially APP epitope-responsive) (Figure 7B). [Figure 7B] Same as above [Figure 8] Figure 8 shows a general model of the effects of CD8+TRM on the brain. [Figure 9A]Figures 9A to 9C show CD8+TRM gene expression in human patient blood (Figures 9A and 9B) and hiTRM staining evaluation indices in human patient blood (by flow cytometry) (in Figure 9C, the columns appear in the order defined from top to bottom in the upper right corner of the figure). [Figure 9B] Same as above [Figure 9C] Same as above [Figure 10A] Figures 10A–10E show abnormal aging T cell gene expression in the blood of normal aging human subjects and human patients with Alzheimer's disease (gene expression omnibus dataset GSE85426). Figures 10A–10C show that three important genes, CD103, CD8A, and CD44, are significantly elevated in Alzheimer's disease, respectively. To ensure that the predictive power is T cell-dependent, patients with below-average expression of the common T cell gene CD3D (Figure 10D) were excluded from the biomarker analysis. Patients younger than 65 years of age (Figure 10E) were also excluded to filter out rare early-onset Alzheimer's disease (AD) with other genetic causes. A P value of less than 0.05(*) indicates a statistically significant difference between normal and AD patients, and **** indicates P<0.0005. [Figure 10B] Same as above [Figure 10C] Same as above [Figure 10D] Same as above [Figure 10E] Same as above [Figure 11A]Figures 11A and 11B show the predictive rates of true and false positives for Alzheimer's disease (AD) using a three-gene panel (CD8, CD44, CD103). After exclusion (as described in the previous paragraph), 40 normal and 49 AD samples remained for biomarker analysis in the high-T cell group (Figure 11A), and 40 samples and 39 AD samples remained for biomarker analysis in the low-T cell group (Figure 11B). The accurate prediction of at least 40% of AD patients, including less than 5% false positive predictions in the high-T cell samples, confirms that abnormal aging T cells are associated with AD and function as a powerful blood biomarker for this late-onset disease. Including CD8A and CD44 along with CD103 in receiver operational characteristics (ROC) analysis allows for more specific identification of the CD8+TRM subpopulation than CD103 alone. Compared to the respective ROC curves based solely on CD103 shown in Figures 12A and 12B, the ROC curves in Figures 11A and 11B are substantially (or significantly) identical, indicating that CD103 is also highly specific to this subpopulation. The slightly lower false-positive rate in this three-gene panel suggests that its value as a biomarker can be further improved by increasing the specificity of TRM. [Figure 11B] Same as above [Figure 12A] Figures 12A and 12B show the predictive rates of true and false positives for Alzheimer's disease (AD) based solely on CD103 from specimens that underwent the same exclusions described for Figures 11D and 11E, for plotting high T-cell groups (Figure 12A) and low T-cell groups (Figure 12B). The accurate predictions of over 50% of AD patients, including less than 8% false positive predictions in high T-cell specimens, confirm that CD103 on T cells is associated with AD and functions as a single-gene blood biomarker for this late-onset disease. [Figure 12B] Same as above [Modes for carrying out the invention]

[0020] All references cited herein are incorporated by reference in their entirety as if they were fully cited. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the field to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology, 3rd edition, revised, J. Wiley & Sons (New York, NY, 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 7th edition, J. Wiley & Sons (New York, NY, 2013); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Press (Cold Spring Harbor, NY, 2012) provide general guidance to those skilled in the art regarding many of the terms used in this application. References for antibody preparation methods include: D. Lane, Antibodies: A Laboratory Manual, 2nd edition (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2013); Kohler and Milstein, (1976) Eur. J. Immunol., Vol. 6: p. 511, U.S. Patent No. 5585089 by Queen et al.; Riechmann et al., Nature, Vol. 332: p. 323 (1988), U.S. Patent No. 4946778; Bird, Science, Vol. 242: pp. 423-442 (1988); and Huston et al., Proc. Natl. Acad. Sci. See also: USA, Vol. 85: pp. 5879-5883 (1988), by Ward et al.; Nature, Vol. 334: pp. 544-554 (1989), by Tomlinson I. and Holliger P.; Methods Enzymol, Vol. 326: pp. 461-479, by Holliger P.; Nat. Biotechnol., September, Vol. 23 (No. 9): pp. 1126-1136.

[0021] Those skilled in the art will understand that there are many methods and materials similar or equivalent to those described herein that can be used in carrying out the present invention. In practice, the present invention is by no means limited to the methods and materials described herein. For the purposes of the present invention, the following terms are defined below.

[0022] T cells can be distinguished from other types of lymphocytes, such as B cells, by the presence of special receptors on the surface of the T cell called T cell receptors (TCRs). Several different T cell subpopulations have been discovered, including T helper cells (T H cells), cytotoxic T cells (T C T cells (CTLs), central memory T cells (T CM T cells) and effector memory T cells (T EM Memory T cells (T cells) M T cells, natural killer T cells (NKT cells), gamma delta T cells (γδT cells), and regulatory T cells (T reg Each of these, including cells, has a different function.

[0023] CD8+ T cells express the CD8 glycoprotein on their cell surface. Most T C Cells (also known as CD8+ T cells) express a TCR that recognizes specific antigens. Antigens within cells (usually peptides produced by the intracellular degradation of proteins) form complexes with MHC class I molecules and are then transported to the cell surface along with these MHC class I molecules, where they are recognized as T cells. C It becomes recognizable by cells. C If the TCR of a cell is specific to that antigen, then the T C The cell binds to its MHC molecule and its peptide complex, and its T C The cells destroy the aforementioned cells. Due to the affinity between CD8 and MHC molecules, during this antigen-specific activation, T C A close connection between the cell and the target cell is maintained. When CD8+ T cells are activated, their partners become T CThey are recognized as cells and are generally classified as having a defined role in cytotoxicity within the immune system.

[0024] As used herein, "APP" refers to amyloid precursor protein. As used herein, "APP peptide" refers to a peptide containing a portion of the APP amino acid sequence. These peptides may be 2 to 20 amino acid lengths (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid lengths). In other embodiments, an APP peptide suitable for use in relation to the aspects of the present invention described herein may be derived from human APP having the full-length sequence shown in Sequence ID No. 1. In one embodiment, the APP peptide includes the sequences ALENYITAL (SEQ ID NO: 2), KLVFFAEDV (SEQ ID NO: 3), LMVGGVVIA (SEQ ID NO: 4), GLMVGGVVI (SEQ ID NO: 5), VIVITLVML (SEQ ID NO: 6), RLALENYIT (SEQ ID NO: 7; amino acids 470-478 of APP), or LALENYITA (SEQ ID NO: 8; amino acids 471-479 of APP). Further description of APP or APP peptides is provided herein by reference in International Patent Publication No. 2017 / 040594. In some embodiments, SEQ ID NOs 2-6 represent readily available APP-derived peptides that can stably bind to the most common HLA allele in the West (HLA-A2), and other peptide and HLA combinations are also available depending on the anthropological characteristics of the patient cohort, as those skilled in the art will understand.

[0025] As used herein, “amino acids” include both natural and synthetic amino acids, and both D-type and L-type amino acids. “Standard amino acids” means any of the 20 L-type amino acids commonly found in natural peptides. “Non-standard amino acids” means any amino acid other than standard amino acids, regardless of whether they are synthetic or of natural origin. As used herein, “synthetic amino acids” also include, but are not limited to, chemically modified amino acids, including salts, amino acid derivatives (amides, etc.), and substituted forms. Amino acids contained in the peptides disclosed herein, particularly those at the carboxyl or amino terminus, can be modified by methylation, amidation, acetylation, or substitution with other chemical groups, which can alter the cyclic half-life of the peptides without adversely affecting their biological activity. Furthermore, the peptides disclosed herein may or may not contain disulfide bonds.

[0026] In this specification, “peptide” and “protein” are used interchangeably and refer to a compound (e.g., a peptide isoster) consisting of at least two amino acid residues covalently linked by a peptide bond or a modified peptide bond. There is no limit to the maximum number of amino acids that can constitute a protein or peptide. The amino acids constituting the peptides or proteins described herein and in the appended claims are understood to be either D-type amino acids or L-type amino acids, with L-type amino acids being preferred. The amino acids constituting the peptides or proteins described herein may be modified by natural processes such as post-translational processing, or by chemical modification techniques well known in the art. Modifications may occur at any site in the peptide, including the peptide backbone, amino acid side chains, and amino-terminus or carboxyl-terminus. It is understood that several sites in a given peptide may have the same type of modification to similar or different degrees. Furthermore, a given peptide may contain multiple types of modifications. Modifications include amino acid additions to transfer RNA-mediated proteins such as acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotide derivatives or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolysis, phosphorylation, prenylation, racemization, selenization, sulfation, arginine addition, and ubiquitination.For example, see Proteins--Structure and Molecular Properties, 2nd edition, by T.E. Creighton, WH Freeman and Company, New York, 1993; and Posttranslational Covalent Modification of Proteins, edited by BC Johnson, Academic Press, New York, 1983, specifically Wold F., "Posttranslational Protein Modifications: Perspectives and Prospects," pp. 1-12; Seifter et al., "Analysis for protein modifications and nonprotein cofactors," Meth. Enzymol., (1990), Vol. 182: pp. 626-646; and Rattan et al., (1992), "Protein Synthesis: Posttranslational Modifications and Aging," Ann NY Acad Sci, Vol. 663: pp. 48-62.

[0027] As used herein, "sample" or "biological sample" refers to tissue or bodily fluids taken from a mammal (preferably a human), and contains CD8 + Includes T cells or CD8 +This refers to samples that are thought to contain T cells. Samples may be blood and / or blood fractions, and may include peripheral blood samples such as peripheral blood mononuclear cell (PBMC) samples or blood (e.g., whole blood, plasma, serum), bone marrow cell samples, or cerebrospinal fluid (CSF). Samples may also be biopsy samples of brain tissue. Samples may include, but are not limited to, lymphocytes, thymus, pancreas, eye, heart, liver, nerves, intestines, skin, muscle, cartilage, ligaments, synovial fluid, and / or joints, or any specific tissue / organ of interest. These samples can be obtained from any individual, including healthy individuals or individuals with cells, tissues, and / or organs that are causing an undesirable immune response. Methods for obtaining such samples are well known to those skilled in immunology and medicine. These methods include the collection and processing of blood and blood components using routine techniques, or the acquisition of biopsy samples from bone marrow or other tissues or organs using standard medical techniques.

[0028] T cells are the primary regulators of inflammation throughout the body. Chronic inflammation is increasingly recognized as a significant contributing factor to various human diseases, and misregistration of T cells exacerbates this chronic inflammation. Memory CD8 subsets abnormally proliferate with age and increase in several tissues, including the brain. However, these proliferations occur rarely in aging experimental animals, and their functional consequences are offset by sustained thymic activity.

[0029] Homeostatic proliferation generally depends on the recognition of autoantigens and / or cytokines, and can therefore promote autoimmunity. Abnormal autoreactive CD8+ T cells are thought to contribute to the onset or progression of individual age-related inflammatory diseases.

[0030] This specification aims to overcome the limitations of common experimental rodent models by examining age-related CD44 hi CD123 + CD127 hi KLRG1 + CD103 +Injection into thymus-deficient mice to obtain a constitutive memory phenotype induces homeostatic proliferation of CD8+ T cells. The resulting artificially homeostatically proliferating (hiT) cells not only exhibit changes in signature aging surface markers and TCRVβ chain clonality, but also show reactivity to central nervous system autoantigens, including amyloid precursor protein (APP) and dopachrome tautomerase / Trp-2, enabling neuropathological studies in hiT cell recipients. RM Similar to cells, these artificial homeostatic, proliferative, and resident memories ( hi T RM These cells are abundant in the brain, and surprisingly, in the brain, they cause (i) increased APP cleavage products, (ii) diffuse β-amyloid (Aβ) plaques in the brain, (iii) fibrillary inclusions in nerve cells, (iv) neuroinflammation, and (v) progressive neurological disease symptoms including age-related cognitive impairment, while also causing a decrease in nerve cells, synaptic markers, and brain mass. hi T cells exhibit pro-inflammatory functions that contribute to neurodegeneration and cognitive impairment in nude mice. A reduction in CD8+ T cells in the brain via CD103 deficiency suppresses age-related cognitive decline in immunonormal mice. Furthermore, T cells are found in both the brains of Alzheimer's disease patients and the blood of patients with cognitive impairment. hi T RM The change in epitope specificity suggests the involvement of this epitope in both disease-related and age-related cognitive decline in humans.

[0031] composition In various embodiments, the present invention provides compositions for the prevention or treatment of age-related neurodegeneration, including pathological neurodegeneration. These compositions include CD103 inhibitors, CD8+T RM This includes effector molecule inhibitors and / or immunotolerogenic vaccines. As used herein, “prevention” includes, but is not limited to, reducing the likelihood of having the above-mentioned disease or condition, or delaying its onset.

[0032] CD103 is also known as integrin αE, and in humans it is encoded by the ITGAE gene, and is also integrin α E CD103 is an integrin protein that is the α subunit of β7 (also known as CD103). CD103 is a tissue-resident memory T(T) protein that is stably present in tissues such as the lungs, intestines, and peripheral non-lymphatic tissues including the skin. RM ) A subtype of memory called CD8 cells + By limiting T cells to specific locations, those cells organize a highly protective local immune response against persistent viral infections.

[0033] In some embodiments, the CD103 inhibitor is an anti-CD103 antibody or an antigen-binding fragment of that antibody. Examples of anti-CD103 antibodies for the prevention or treatment of neurodegeneration include (1) PE anti-human CD103 antibody (BIOLEGEND®) derived from clone Ber-ACT8, (2) mouse anti-human CD103 monoclonal antibody (mAb) (BIORAD®) derived from clone 2G5.1 or a humanized antibody of 2G5.1, (3) OX-62 or a humanized antibody of OX-62, which is an anti-rat CD103 monoclonal antibody (mAb), and (4) anti-mouse CD103 monoclonal antibody (EBIOSCIENCE®) derived from clone 2E7 or a humanized antibody of 2E7.

[0034] In other embodiments, the CD103 inhibitor is CD8+T RM In yet another embodiment, the CD103 inhibitor is a small molecule that inhibits the activity of CD103. In yet another embodiment, the CD103 inhibitor is a nucleic acid that silences or cleaves the DNA or mRNA corresponding to CD103. In yet another embodiment, the CD103 inhibitor is paxilin, which is a protein that binds to at least the cytoplasmic domain of CD103.

[0035] In some embodiments, CD8+T RM Effector molecule inhibitors are administered to promote the treatment, suppression, reduction of the severity, or prevention of age-related cognitive decline, pathological neurodegeneration, or both. These effector molecule inhibitors are APP-specific CD8+TRM The present invention relates to small molecules, antibodies, antibody fragments, or nucleic acids that inhibit the activity of perforin 1, interferon-gamma, or other inflammatory cytokines released by converted effector T cells, or that reduce their expression levels. In one embodiment, a perforin 1 inhibitor is administered, which includes, but is not limited to, diarylthiophene and GSK2126458. In another embodiment, an interferon-gamma (IFNγ) inhibitor is administered, which includes, but is not limited to, mesopram and locaglamide.

[0036] In yet another embodiment, the immunotolerogenic vaccine includes a vaccine that delivers an effective amount of amyloid precursor protein or its peptide, which includes, but is not limited to, the peptides of SEQ ID NOs: 2-8.

[0037] Pharmaceutical composition In various embodiments, the present invention provides pharmaceutical compositions for the prevention or treatment of age-related neurodegeneration. These pharmaceutical compositions include CD103 inhibitors, CD8+T RM The present invention includes an effector molecule inhibitor (e.g., perforin 1 inhibitor and IFNγ inhibitor), and / or an immunotolerogenic vaccine, and a pharmaceutically acceptable excipient. In one embodiment, the CD103 inhibitor is an anti-CD103 antibody. In another embodiment, the effector molecule includes perforin, interferon-γ, or other inflammatory cytokines. In yet another embodiment, the immunotolerogenic vaccine includes APP, or APP peptides such as the peptides of SEQ ID NOs. 2-8.

[0038] The pharmaceutical compositions of the present invention may contain any pharmaceutically acceptable excipients. “pharmaceutically acceptable excipients” generally means excipients that are safe, non-toxic, and useful in preparing desirable pharmaceutical compositions, and include excipients that are acceptable for use in human pharmaceuticals and similarly acceptable in veterinary medicine. Such excipients may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gas. Examples of excipients include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulators, lubricants, binders, disintegrants, wetting agents, emulsifiers, colorants, release agents, coatings, sweeteners, flavoring agents, fragrances, preservatives, antioxidants, plasticizers, gelling agents, thickeners, curing agents, setting agents, suspending agents, surfactants, water-retaining agents, carriers, stabilizers, and combinations thereof.

[0039] In various embodiments, the pharmaceutical compositions of the present invention may be formulated for delivery via any route of administration. “Route of administration” may be any route of administration known in the art, including, but not limited to, aerosol, nasal, oral, mucosal, dermal, parenteral, or intestinal. “Pareral” refers to routes of administration generally associated with injection, including intraorbital, intrainfusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intramedullary, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, mucosal, or transtracheal. When delivered via a parenteral route, the composition may be in the form of a solution or suspension for intravenous or infusion, or a lyophilized powder. When administered via the intestinal route, the pharmaceutical composition may be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres, or lipid vesicles or polymer vesicles, allowing for controlled release. These compositions are typically administered by injection. These methods of administration are known to those skilled in the art.

[0040] The pharmaceutical compositions according to the present invention may include any pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle that is involved in the transport or delivery of the compound of interest from one tissue, organ, or body part to another. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other components of the formulation. The carrier must also be suitable for use in contact with any tissue or organ it may come into contact with, meaning that the carrier must not carry any risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that significantly outweighs the therapeutic benefits of the carrier.

[0041] The pharmaceutical compositions according to the present invention can be formulated as capsules, tablets, or suspensions or syrups for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the compositions, or to facilitate their preparation. Examples of liquid carriers include syrup, peanut oil, olive oil, glycerin, saline solution, alcohol, and water. Examples of solid carriers include starch, lactose, calcium sulfate dihydrate, clay, magnesium stearate or stearic acid, talc, pectin, acacia gum, agar, or gelatin. The carriers may contain sustained-release substances (e.g., glyceryl monostearate or glyceryl distearate) alone or together with wax.

[0042] Pharmaceutical preparations are prepared according to conventional pharmaceutical techniques, involving, as necessary, crushing, mixing, granulation, and tableting in the case of tablet dosage forms, or crushing, mixing, and filling in the case of hard gelatin capsule dosage forms. When liquid carriers are used, the preparations are in the form of syrups, elixirs, emulsions, or aqueous or non-aqueous suspensions. Such liquid preparations may be administered orally as is, or filled into soft gelatin capsules for administration.

[0043] The pharmaceutical composition according to the present invention may be delivered in a therapeutically effective dose. This precise therapeutically effective dose is the amount of composition that produces the most effective result in terms of therapeutic efficacy in a given subject. This amount varies depending on various factors, including (but not limited to) the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological state of the subject (including age, sex, type and stage of disease, general health status, responsiveness to a given dose, and type of drug), the properties of the pharmaceutically acceptable carrier or carrier in the formulation, and the route of administration. Those skilled in the art in the medical and pharmacological fields can determine the therapeutically effective dose through routine experiments, for example, by checking the subject's response to the administration of the compound and adjusting the dose accordingly. For further guidance, see Remington, The Science and Practice of Pharmacy (edited by Gennaro, 20th edition, Williams & Wilkins, Pennsylvania, USA) (2000).

[0044] Another drug delivery system for increasing the circulating half-life is liposomes. Methods for preparing liposome delivery systems have been discussed in Gabizon et al., Cancer Research (1982), Vol. 42: p. 4734; Cafiso, Biochem Biophys Acta (1981), Vol. 649: p. 129; and Szoka, Ann Rev Biophys Eng (1980), Vol. 9: p. 467. Other drug delivery systems are known in the art and are described, for example, in Poznansky et al., DRUG DELIVERY SYSTEMS (ed. RL Juliano, Oxford, New York, 1980), pp. 253-315; and ML Poznansky, Pharm Revs (1984), Vol. 36: p. 277.

[0045] After the preparation of a liquid pharmaceutical composition, it may be freeze-dried to prevent degradation and maintain sterility. Methods for freeze-drying liquid compositions are known to those skilled in the art. The composition may be reconstituted immediately before use with a sterile diluent (e.g., Ringer's solution, distilled water, or sterile saline), which may contain additional components. The reconstituted composition is then administered to the target using methods known to those skilled in the art.

[0046] kit In one embodiment, the kit extracts CD103-positive CD8+T from a biological sample. RM Identify the group, It includes components necessary for isolation and / or concentration. In another embodiment of this embodiment, the kit may further include positive controls and / or negative controls and / or instructions for identifying, isolating, and / or concentrating CD103-positive CD8+ T cells using the contents of this kit. In yet another embodiment of this embodiment, the kit may further include culture vessels (e.g., dishes or flasks), culture media, or any necessary buffers and factors useful for promoting cell growth.

[0047] In another embodiment, the kit is used to obtain CD8A-positive, CD44-positive, and CD103-positive CD8+T from a biological sample. RM It includes components necessary for identifying, isolating, and / or concentrating populations. In another embodiment of this embodiment, the kit may further include positive controls and / or negative controls and / or instructions for identifying, isolating, and / or concentrating CD8A-positive, CD44-positive, and CD103-positive CD8+ T cells using the contents of this kit. In yet another embodiment of this embodiment, the kit may further include culture vessels (e.g., dishes or flasks), culture media, or any necessary buffers and factors useful for promoting cell growth.

[0048] Instructions for use may be included with this kit. These instructions typically contain clear language describing the techniques used in using the components of this kit to achieve desired outcomes, such as the treatment, reduction, suppression, or prevention of age-related neurodegeneration. Optionally, the kit may also include other useful components, such as measuring devices, diluents, buffers, pharmaceutically acceptable carriers, syringes, or other useful tools readily understood by those skilled in the art.

[0049] Various embodiments have been found to have CD8A-positive, CD44-positive, and CD103-positive CD8+T RM Detection or CD8+T RM The detection of other biomarkers mentioned above is performed using flow cytometry analysis based on the fluorescence-activated cell sorting (FACS) technique. Detailed standard operating procedures for FACS flow cytometry analysis are presented in Example 2.

[0050] How to use A method for identifying individuals who are prone to or already have pathological neurodegeneration is to examine the peripheral blood of those individuals for CD103-positive resident memory CD8+ T cells (CD8+ T RM This includes detecting an increase in the presence of CD8+T10A, CD44, and CD103 in the blood. In other embodiments, the subjects are human subjects aged at least 65 years and have CD8+T10A, CD44, and CD103 positive in their blood. RM An increase in the level is detected.

[0051] CD103-positive CD8+T in subjects with memory impairment or age-related neurodegeneration RM A method for quantifying CD103-positive CD8+T in a biological sample derived from the above subject, wherein CD103-positive CD8+T RM Methods are also provided that include detecting the quantity of cells. In some embodiments, this method is used to detect CD103-positive CD8+T RM This further includes comparing the cell count to a baseline value.

[0052] Various detection methods are available in biological samples, including, but not limited to, flow cytometry, Western blotting analysis, enzyme-linked immunosorbent assays, immunoprecipitation, UV spectrophotometering, chromatography, mass spectrometry, immunohistochemical staining, and imaging.

[0053] The reference value for a quantitative assay or quantitative method may be a value obtained from a single control subject (e.g., a healthy subject with no symptoms of memory impairment or neurodegeneration) or a value obtained from a pool of multiple such control subjects. In another embodiment, the reference value is the subject's own value at a young age when there are no or very few symptoms of memory impairment, and if the current value exceeds that reference value, it is used as a criterion for determining high risk, the need for treatment of the symptoms, or suboptimal treatment outcomes. In yet another embodiment, the reference value is the subject's own value before treatment for memory impairment or neurodegeneration, and is used as a criterion for determining the efficacy of the treatment.

[0054] Methods are provided to treat, suppress, reduce the severity of, or promote the prevention of age-related cognitive decline, pathological neurodegeneration, or both, in the target population. These methods involve CD103 inhibitors, resident memory CD8+ T cells (CD8+ T RM Effector T cell inhibitors derived from ) and CD8+T RM The method includes administering to the subject one or more therapeutically effective doses of inhibitors of molecules released from the effector T cells resulting from the above. In some embodiments, the CD103 inhibitor in this method is an anti-CD103 antibody, and the inhibitor of effector T cells resulting from resident memory CD8+ T cells includes a perforin 1 inhibitor or an IFNγ inhibitor. According to one embodiment, this administration induces CD8+ T to APP peptide (e.g., the peptide of SEQ ID NO: 8). RMOr the response or binding of effector T cells derived therefrom is reduced compared to that obtained in one control subject (e.g., one healthy subject without any symptoms of memory impairment or neurodegeneration) or compared to that obtained from a pool of multiple such control subjects. In another embodiment, this administration reduces CD8+ T cells to APP peptide (e.g., the peptide of SEQ ID NO: 8). RM Alternatively, the response or binding of effector T cells derived therefrom is reduced compared to the subject's own values ​​at a younger age when there are no symptoms of memory impairment or very few symptoms of memory impairment.

[0055] Methods are also provided for treating, suppressing, reducing the severity of, or promoting the prevention of age-related cognitive decline, pathological neurodegeneration, or both in subjects requiring treatment. These methods include administering a therapeutically effective dose of an immunotolerative vaccine that delivers an amyloid precursor protein or a peptide fragment thereof (e.g., any of the peptides of SEQ ID NOs. 2-8) to the subjects.

[0056] A method for identifying human subjects who are prone to or have age-related cognitive decline or pathological neurodegeneration, comprising identifying CD103+ resident memory CD8+ T cells (CD8+ T) in blood samples obtained from human subjects who have one or more symptoms among short-term or long-term memory loss, decreased ability to maintain concentration, and decreased ability to solve problems. RM A method is provided which includes detecting an increase in the presence of ). According to one aspect of this method, CD103+CD8+T RM The increased presence of is compared to a value obtained from a pool of one or more healthy human subjects who do not have any of the above one or more symptoms. In another embodiment, this human subject is at least 65 years old, or at least 50, 55, or 60 years old.

[0057] In various embodiments, the subject in the above method is a human. In some embodiments, the human subject is middle-aged or older, for example, 30 years or older, 35 years or older, 40 years or older, 45 years or older, 50 years or older, 55 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, 90 years or older, or 95 years or older. In other embodiments, the human subject has previously shown a record of CD103-positive CD8+T RM cells.

[0058] In various embodiments, age-related cognitive impairment, pathological neurodegeneration, or memory impairment in one or more of the above methods and compositions includes symptoms such as amnesia or loss of short-term or long-term memory, reduced ability to maintain concentration, reduced problem-solving ability, multiple sclerosis, Parkinson's disease, and Alzheimer's disease.

[0059] such as animal models A system for identifying and / or screening candidate therapeutic, preventive, and / or diagnostic agents for human cognitive decline, comprising CD44 obtained from a rodent (e.g., mouse) hi CD123 + CD127 hi KLRG1 + CD103 + The above system including the phenotype of resident memory CD8+T cells is provided. In some embodiments, this CD44 hi CD123 + CD127 hi KLRG1 + CD103 + The resident memory CD8+T cell phenotype is obtained by administering resident memory CD8+T cells to thymus-deficient mice.

[0060] A method for identifying and / or screening candidate agents for the treatment or prevention of age-related neurodegeneration in humans comprises exposing a candidate agent in vitro to CD44 hi CD123 + CD127 hi KLRG1 + CD103 +Contact with resident memory CD8+ T cells, or CD44 hi CD123 + CD127 hi KLRG1 + CD103 + This involves administering a candidate drug to an animal model containing resident memory CD8+ T cells to identify the level of reduction in CD103-positive resident memory CD8+ T cells, the level of reduction in effector molecules of these cells, or the reduction in the migration of CD8+ T cells from the peripheral system to the brain of the animal. [Examples]

[0061] The following examples are provided to better illustrate the invention claimed in this application and should not be construed as limiting the scope of the invention. Any references to specific materials are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can develop equivalent methods or reactants without inventiveness and without departing from the scope of the invention.

[0062] Example 1. Abnormal commensal memory CD8+ T cells lead to pathological neurodegeneration and age-related cognitive decline. CD8+ T cell homeostatic proliferation is a function of a small number of T cells, and it occurs not only gradually with age but also rapidly when injected into young T cell-deficient hosts (20, 21). Given that this phenomenon has been proven to be associated with age-related CD8+ T cell dysfunction, this induceable phenomenon has made it possible to clearly examine the role of abnormal CD8+ T cells in diseases such as Alzheimer's disease. Spontaneous induction of homeostasis by injection into nude mice was found to uniformly induce CD8+ T ("hiT") cells exhibiting molecular abnormalities, phenotypic abnormalities, and functional abnormalities indistinguishable from those in diseased aging mice. These hiT cells localize in the brain, where they ultimately promote Alzheimer's disease-like neurodegenerative symptoms, including prominent disease features lost in FAD mutant gene transgenic animals. The hiT cell-related rating index was also elevated in the brains of human Alzheimer's disease patients. Our research has identified age-related immune cell processes that overcome resistance in mice to age- and risk-factor-induced Alzheimer's disease-like symptoms. These findings have significant implications for modeling age-related diseases in mice, as well as for modeling sporadic Alzheimer's disease, investigating its etiology, and developing treatments.

[0063] Materials and methods Animals Female C57BL / 6, B6.Foxn1 mice, and related and / or identical knockout lines (Jackson Laboratories) were freely fed in a pathogen-free animal housing facility under standard conditions of a 12-hour light / 12-hour dark cycle. Recipient animals were 8-10 week old female B6.Foxn1 mice (n>5), B6.Foxn1-AppKO mice (n>4), or B6.CD45.1 related mice (n>5), with donors being 5-8 week old females of the same lineage. Cell sources were randomized by pooling more than 5 donors per experiment. Age-related cognitive decline was studied using young (8-10 weeks) and older (15 months) male and female C57BL / 6 and B6.CD103-knockout mice (n=12 for young mice and n=7-8 for older mice). Donor animals, recipient animals, and untreated animals were housed in the pathogen-free facility of the Department of Comparative Medicine at Cedars-Sinai Medical Center, and all breeding and genetic screening were performed at Jackson Laboratories (Bar Harbor, Maine).

[0064] Adoptive transfer of CD8+ T cells Spleen CD8+ T cells from female C57BL / 6J mice (5-7 weeks old) were purified using anti-CD8 immunobeads (Milteny Biotech, Sunnyvale, California). 3 × 10⁶ cells were extracted in 50 μl of PBS. 6 Individual CD8+ T cells were intravenously injected into female C57BL / 6J hosts or B6.Foxn1 nude mouse hosts. The efficiency of introduction into the B6.Foxn1 host was verified by the colonization of more than 5% of CD8+ T cells in splenic lymphocytes three weeks after injection. The order of treatment was randomized by alternating between cell injection and control injection among individual recipients. For all subsequent analyses, both the identity of the groups and the expected results were concealed from the researchers performing the analyses.

[0065] Processing of tissues (brain, spleen) Brains and spleens were collected from PBS-perfused mice. 1 mm sections of the brain were prepared up to the right side of the longitudinal fissure (midline). For protein studies, the right hemisphere was flash-frozen at -80°C, followed by homogenization in cell lysis buffer (Cell Signaling Technologies, Massachusetts), and the cell nuclei were centrifuged. Cell lysates were separated into triton-soluble, sarcosyl-soluble, and sarcosyl-insoluble fractions using sequential incubation of 10% (g / vol) salt sucrose solution and 1% (g / vol) sarcosyl salt sucrose solution. The left hemisphere was fixed in 4% paraformaldehyde and preserved for immunohistochemical staining. Brain weight standardization: The entire brain was removed from the skull, and the cerebellum, brainstem, and olfactory bulb were removed before weighing on a Mettler balance.

[0066] Western blot Triton-soluble cell fractions were separated by electrophoresis on a 12% Tris-HCl precast gel (Bio-Rad) and blotted onto 0.2 μm nitrocellulose. The membranes were blocked with BSA and incubated continuously for 1 hour at room temperature in dilutions of primary and secondary antibodies, followed by more than three washes. The membranes were then developed using enhanced chemiluminescent substrate (GE Healthcare Biosciences, Pittsburgh, Pennsylvania) and exposed to Amersham Hyperfilm (GE Healthcare Biosciences, Pittsburgh, Pennsylvania).

[0067] ELISA The supernatant of homogenized brain tissue was used as the Triton-soluble fraction of Aβ. Insoluble pellets derived from brain homogenized with Triton were resuspended in 10 times the volume of 5M guanidine hydrochloride for 4 hours to prepare guanidine-soluble Aβ. The Triton-soluble and guanidine-soluble samples were analyzed for soluble and insoluble Aβ using ELISA (Invitrogen, Life Technologies; Grand Island, New York). Absorbance was read using a SPECTRAmax Plus 384 microplate reader (Molecular Devices, Sunnyvale, California), and delta was analyzed using Graphpad PRISM (Graphpad Software, San Diego, California).

[0068] Flow cytometry Purified T cells stained with each antibody were analyzed for purity using tricolor flow cytometry (FACScan II; BD Hyoh Science, San Jose, California). Whole spleen single-cell suspensions and antibodies in PBS containing 5% FBS were incubated on ice for 30 minutes, followed by washing with PBS containing 5% FBS. 100,000–300,000 flow events were acquired.

[0069] Antibodies for tissue staining and Western blotting Free-floating brain sections (8–14 μm thick) were placed on slides and blocked at room temperature for 1 hour. The sections were incubated overnight at 4°C with primary antibody in blocking solution (Dako, California). The sections were washed four times in PBS and incubated for 90 minutes with curcumin (0.01% in PBS) or without curcumin with fluorochrome-conjugated or biotin-conjugated secondary antibody, or with thioflavin S alone (1% in PBS). The sections were washed, covered with coverslips, and placed on ProLongGold anti-bleed media containing DAPI (Invitrogen). Bright-field and fluorescence images were obtained using a Zeiss AxioImager Z1 (Carl Zeiss Microimaging) with a CCD camera. Microscopic image analysis was performed using ImageJ (NIH). Anti-Aβ / APP antibody (Abcam ab14220 for the 3-week mark; Chemicon clone 4G8 for all others) was used at 1:500 for immunohistochemistry (IHC) and 1:1000 for Western blotting (WB). Anti-p-tau pS199 / 202 antibody (Invitrogen) was used at 1:50 for IHC and 1:100 for WB, and phospho-PHF-tau pSer202+Thr205 antibody (AT8) was used at 1:2000 for WB to confirm PHF. For marker size, the WB signal of p-tau was normalized to the signal of β-actin (clone AC-74, Sigma), and GAPDH was used to normalize all other markers. Anti-GFAP (Dako) was used at 1:250 for IHC and WB. Anti-NeuN antibody (Chemicon) was used at 1:100 for IHC and WB. Anti-Iba1 (Wako Corporation) was used in IHC at a 1:200 ratio. Anti-CD8 (clone 53-6.72, BD Phermigen) was used in IHC at a 1:100 ratio and in WB at a 1:1000 ratio. All secondary antibodies (HRP, AlexaFlour-488, -594, -647; Invitrogen) were used in IHC at a 1:200 ratio and in WB at a 1:200 ratio.Multimer construction and use: Dextramers of epitopes constructed for autoantigen / brain antigens (Trp-2-DCT(180-188) / H-2Kb) and / or custom APP epitopes with a predicted affinity of less than 100 nM (NetMHC version 3.4) were constructed using Immudex.

[0070] Galius silver dyeing Fibrillar aggregates were visualized using Gallius silver staining. Free-floating brain sections were placed in 5% periodic acid for 3 minutes, washed twice, placed in silver iodide solution for 1 minute, incubated in 0.5% acetic acid for 5 minutes (twice), and washed with distilled water. The sections were incubated in developer for approximately 10 minutes until they turned light brown / gray, placed in 0.5% acetic acid for 5 minutes to stop color development, washed with distilled water, and placed on glass slides. The stained sections were examined by microscopic observation. Stained neurons were counted from CA2 in the hippocampus, and the number of stained neurons in all neurons of the entorhinal cortex and cingulate cortex was visually quantified in three experiments.

[0071] Counting of nerve cells The total number of neurons was estimated using optical fractionation methods in conjunction with stereoscopic analysis software (Stereo Investigator; MBF Bioscience). Paramedian sagittal serial sections, spaced 50 μm apart, were stained for NeuN. Based on Paxinos and Watson's mouse brain atlases, grids were randomly placed on ROIs (Regions of Interest) limiting CA1, CA2, CA3, and other regions of interest. The number of cells within a three-dimensional optical dissector (50 μm × 50 μm × 10 μm) was counted using a 100x objective lens. A 1 μm guard zone on the top and bottom of the section was excluded within each dissector. Using the Stereo Investigator software, a weighted total number was obtained, with an error coefficient of 0.10.

[0072] Behavioral Test Open-field tests were conducted 3, 6, and 13 months after cell or control injection, prior to all other behavioral tests. The number of flinch jump / fear-conditioned freezing behaviors was determined 6 and 11 months after cell or control injection. Mice were tested for SA only once 12 months after cell or control injection. The Burns maze test was conducted only once 14 months after cell or control injection. The order of behavioral tests was randomized by alternating experiments with the control and treatment groups. For tests lasting more than one day, tests were started simultaneously (±1.5 hours) and early and late tests were alternated for inter-group randomization. In the Burns maze, additional randomization was employed by alternating the location of the escape area among individual animals in each group and among each of the three daily training tests per animal.

[0073] Barnes Maze (BM) Test The Barnes maze is a spatial learning task that allows subjects to use spatial cues to determine a means of escaping from a somewhat aversive environment (i.e., these mice are required to use spatial cues to find a refuge). The ability of mice to learn the location of the escape box was evaluated over a 9-day period in a BM apparatus. The escape hole remained constant for each mouse over a 5-day training period. Each mouse was tested three times daily for 4 days (3 trials), followed by 2 days of no testing, and then retested on day 7. Each trial was divided into 35-60 minute intervals between trials. Each trial began with one mouse placed in a bottomless cube start box located in the center of the maze. After 30 seconds, the start box was lifted, and the mouse was released to find the escape hole. Two fluorescent lights located on the ceiling or high in the room illuminated the test chamber. Each trial lasted a maximum of 4 minutes, or until the mouse entered the escape box. After each training test, the experimenter guided mice that failed to find the escape hole within four minutes to the correct hole. Once the mouse entered the escape box, it was kept inside for one minute. After the seventh day of testing, but on different days, the mice were tested for two more days, in which the escape box was placed in the opposite position on the eighth day and then returned to its original position on the ninth day. The exact same testing method was applied to all mice in all groups. After each test and before each daily test, the maze and all compartments were thoroughly cleaned with isopropyl alcohol to remove any olfactory cues.

[0074] Y-maze spontaneous alternating behavior (SA) test The Y-maze alternation behavior test is used to assess working memory. Spontaneous alternation behavior is measured by individually positioning animals on one arm of a Y-maze made of opaque black acrylic (arm section: 40 cm long, 4 cm wide; wall section: 30 cm high). The order of entry into the arms and the total number of entries over an 8-minute period are recorded. Mice were tested with SA only once.

[0075] Flinch jump / Fear conditioning test First, the flinch-jump test was used to determine if there was a significant difference in nociceptive threshold (pain sensitivity) between the treatment groups. Subsequently, learning and memory of aversive events were evaluated using Pavlovian fear conditioning. The apparatus (Freeze Monitor®, San Diego Instruments, San Diego, California) consisted of a plexiglass box (25.4 × 25.4 × 31.75 cm high) with a stainless steel grid floor. An auditory stimulation unit was placed at the top of this box, and light beams and optical sensors were placed around the box. These optical sensors were connected to a computer via an input matrix to automatically record the interruption of the light beam. For the test, on day 1, individual mice were placed in this test box and allowed to acclimate for 3 minutes. At the 3-minute mark, a sound was played for 30 seconds. 30 seconds after the sound stopped, a 0.5-second foot stimulus (intensity = the average jump threshold of the treatment group determined by the flinch-jump test) was given. The mouse was then removed from the box and returned to its home cage for 2 minutes. The chamber was cleaned, the animal was returned to the chamber, and this procedure was repeated. The number of freezing behaviors (no movement exceeding 5 seconds and no blockage of light beam) during this procedure was recorded using a freezing behavior monitoring device. On the second day, the mouse was placed in the same test box where it had previously received auditory and foot stimuli, to determine if it could recall the situation, but without the auditory and foot stimuli this time. The number of freezing behaviors was measured over 10 minutes. On the third day, cue conditioning was measured after placing a triangular plexiglass box inside the test box. The mouse was placed in this triangular chamber where it had never previously received auditory or foot stimuli, and after 1 minute, it was given a sound for 30 seconds, and the number of freezing behaviors was measured over 10 minutes. All data from the flinch jump and fear conditioning tests were first normalized within each group to the mean of the first two training tests on day 1, then normalized within all experimental groups to the contextually learned freezing behavior or cue-learned freezing behavior values ​​of the PBS control, expressed as a percentage relative to the control, and analyzed by ANOVA, followed by Newman-Coils test where appropriate, to detect differences between treatment groups.

[0076] Open field test This test was carried out in an open field apparatus made of a transparent plexiglass box with an open top, measuring 16” × 16” × 15” high. Two ring-shaped light beams and optical sensors were arranged around this box. These optical sensors were connected to a computer by an input matrix. Each mouse was placed into this box, and the interruption of the light beam was automatically recorded and used as a measure of locomotor activity. Each mouse was tested in this box for a period of 30 minutes.

[0077] Statistical analysis For regions containing the hippocampus and cortex of 900 - 1200 μm, at 150-μm intervals (unless otherwise indicated), the cell number or area (μm 2 ) of β-amyloid plaques, GFAP+, Iba1+, or Perforin1+ cells in 6 - 8 coronal sections from each individual was quantitatively analyzed using stereological counting methods. For each image, specific fluorescence signals were captured at the same exposure time, and optical sections of each field of the specimen were input into NIH ImageJ and analyzed as described above. GraphPad Prism (version 5.0b; San Diego, California, USA) was used for data analysis using ANOVA and Welch's corrected t-test (not assuming equal variance). Mean + SEM is shown in all histograms.

[0078] The sample sizes of the PrfKO-CD8 group and the IfnγKO-CD8 group were calculated a priori for each evaluation index using the mean values and standard deviations of the PBS group and the wild-type CD8 group for the expected effect size, with an α of 0.05 and a confidence level exceeding 95%. Then, a number greater than 1 was added to the calculated n and used for the PrfKO-CD8 group and the IfnγKO-CD8 group.

[0079] Sections or samples that do not contain a recognizable background signal, and values ​​within each group that are above or below twice the standard deviation from the median of each group, are included in those predetermined to be excluded. The number of subjects and the reagent validation method are described in Table S1.

[0080] Research approval All animal experimentation procedures were approved by the Animal Experimentation Board at Cedars-Sinai Medical Center prior to execution. The Cedars-Sinai Medical Center Institutional Review Board (IRB) designated the analysis of anonymized human brain specimens originating from the University of California, Davis, as exempt from its review. The brain specimens were collected, stored, and distributed under prior approval by the IRB at the University of California, Davis Medical Center.

[0081] result Generation of "hiT" cells in nude mice CD8+ T cells derived from young (less than 9 weeks old) C57BL / B6 (B6) donors were injected into B6.Foxn1 recipients and subjected to phenotypic analysis (Figures 1A, 1H, and 1I). Donor CD8+ T cells rapidly proliferated in the blood of young B6.Foxn1 recipients within 3 days, and these cells remained in the blood for a long period (Figures 1J and 1K). From nude mouse hosts to wild-type B6 hosts or B6.CD45.2 gene-derived [B6] (Cg) CD8+ T cells that were continuously transferred into the host did not proliferate further (Figure 1I and Figure 1K). B6. Analysis of artificially homeostatically proliferating donor CD8+ T cells ("hiT" cells) in the Foxn1 host showed the same surface marker profile as CD8+ T cells that underwent clonal proliferation in aged mice (CD122). hi CD127 hi CD44 hi KLRG1 hi PNA hi CD8 lo CD103 +(Figures 1A-1D). A similar phenotype is observed in the clonal proliferation of CD8+ T cells in aging humans. Therefore, CFSE-labeled CD8+ T cells showed a ladder-like pigment dilution and population expansion typical of homeostatic proliferation (Figure 1K). However, this did not occur in nude mice lacking the amyloid precursor protein (APP) gene (B6.Foxn1xAppKO mice), suggesting that rapid homeostatic proliferation may be dependent on responsiveness to APP.

[0082] To investigate the clonality of hiT cells, we analyzed the D→J rearrangement of the variable region in the T cell receptor β gene segment by PCR. Consistent with previous reports, peripheral T cells in 12-month-old wild-type mice showed no evidence of clonal skew in the TCRVβ chain, but peripheral T cells injected into nude mouse recipients showed clonal skew of D1→J1 and D2→J2 just 10 weeks later (Figures 2F and 2G). Importantly, in contrast to the diverse D→J utilization observed in young wild-type mice, the D1→J1 and D2→J2 clonal skew was also evident in the brains of young nude mice injected with CD8+ T cells (Figures 1E and 1F). This pattern most closely resembled D→J utilization in the brains of aged mice.

[0083] CFSE-labeled donor CD8+ T cells increased in the brain parenchyma of B6.Foxn1 3 days after intravenous infusion, directly demonstrating the rapid return of hiT cells to the brain (Figures 2A and 2B). Flow cytometry showed only a slight increase in total CD8+ T cells in B6.Foxn1 10 weeks after infusion compared to wild-type B6 (Figure 2C), but Western blotting clearly showed an increase in CD8 protein at this point, suggesting increased cell influx without an increase in viable cells (Figure 2H). Indeed, flow cytometry revealed that IFNγ retained CD103 expression. + CD8+ T cells and KLRG1 +Both CD8+ T cells were significantly increased in the brains of nude mouse recipients at this point, indicating a qualitative rather than quantitative change in CD8+ T cells in the brain (Figure 2C). KLRG1 in peripheral blood + CD8+ T cells respond to MHC class I restriction antigens, including tyrosinase-related protein-2 / dopachrome tautomerase (Trp-2 / DCT) and APP, but only the latter was significantly increased in the brain (Figures 2D and 2E). Thus, since hiT cells responsive to the APP epitope selectively accumulated in the brains of nude mice, the inventors decided to analyze APP-related symptoms (Figure 3K).

[0084] Aβ and neurofibrillary deposits Western blotting revealed that surfactant-soluble APP and derived cleavage products (APP) were present in excised corticosteroids and hippocampi of B6.Foxn1 hosts 3 and 10 weeks after intravenous CD8+ T cell infusion. Cl ) increased (Figures 3A and 2I). Aβ1-40 increased at 2.5 months according to ELISA and remained elevated at 15 months (Figure 3B), and increased Aβ in the vascular system was observed at 6 months (Figures 3L and 3M). Diffuse plaques and increased Aβ1-40 were detected in the hippocampus, entorhinal cortex, and cingulate cortex of B6.Foxn1 recipients (wild-type CD8 group mice) injected with wild-type CD8+ T cells at 15 months (Figures 3C and 3N). However, unlike mice expressing familial gene mutations seen in human Alzheimer's disease, Aβ1-42 did not change much in hiT-carrying nude mice, amyloid plaques were mainly diffuse and hardly co-stained with curcumin or thioflavin S (Figures 3C and 3O). Therefore, amyloidopathy in hiT-carrying nude mice differed from amyloidopathy seen in the ADtg mouse model.

[0085] Cells in the dentate gyrus of wild-type CD8 mice 6 months after T cell injection were stained with curcumin and thioflavin S (Figure 3K). Similar structures were not observed in aged ADtg mice (Figure 3K) or ADtg rats exhibiting tau-paired spiral fibrils (Figure 3L). This suggests that hiT-carrying nude mice may possess fibrous inclusions composed of highly phosphorylated tau protein in their neurons. Consequently, in the brains of wild-type CD8-carrying mice 10 weeks after injection, p-tau in the triton-soluble fraction increased by approximately 30%, and greater tau PHF increased nearly 5-fold (Figures 3E, 3F). Although the increase in p-tau was not sustained, tau PHF remained 2.5-fold higher than in controls 15 months after injection (Figure 3F). Most interestingly, silver-stained cells were also increased at this point in the hippocampus, entorhinal cortex, and cingulate cortex of wild-type CD8 mice (Figure 3G, Figure 3H). Serial silver / immunofluorescence staining revealed these to be nucleated p-tau cells. + Although they were shown to originate from nerve cells, anucleated "ghost tangles" were not observed (Figures 3G, 3M, and 3N). The brains of ADtg mice stained simultaneously showed only silver-stained plaques (Tg2576 mice; Figure 3G), thus confirming that silver-stained cells were only found in hiT-carrying mice. These data suggest that hiT cells promote histological deposition of Aβ40 in the parenchyma, diffuse plaques, and fibrillary inclusions in living nerve cells.

[0086] Immune and neuroinflammatory infiltration Although not evident from forebrain flow cytometry, the number of CD8+ T cells was significantly increased in hippocampal sections of wild-type CD8 mice 15 months after injection, and these cells happened to be p-tau + It interacted with nerve cells (Figure 3O and Figure 3P). CD8+ T cell counts did not increase in the lateral hippocampus. Iba1 in the cortex and hippocampus + Microglia and Activated GFAP +Astrocytes were also significantly increased in wild-type CD8 mice compared to controls (Figures 3I and 3J). The Aβ plaque area ratio correlated more strongly with hippocampal CD8+ T cell count than with cortical or hippocampal astroglia proliferation, consistent with the strong influence of T cells on amyloidopathy (Figures 3Q, 3R, and 3S).

[0087] Loss of nerve cells and brain atrophy Fifteen months after T cell injection, NeuN in CA2 cells was observed in wild-type CD8 mice compared to controls. + Cell counts decreased (Figures 4A-4C). Furthermore, brain volume decreased by 5% in the wild-type CD8 group 6 months after T cell injection, progressing to a 10% decrease by 15 months (Figure 4D). Significant neuronal and synaptic loss in the wild-type CD8 group was confirmed by decreased NeuN signaling, drebrin signaling, and synaptophysin signaling in Western blots, each showing approximately a 10% decrease in signaling at 15 months after injection (Figures 4E and 4F). Western blot signaling for NeuN was significantly correlated with brain volume between treatment groups, indicating that brain atrophy reflects neuronal loss (Figure 4G).

[0088] Severe cognitive impairment Overall motor and rearing activity did not differ significantly between the treated and control nude mouse recipients at 3, 6, or 13 months after T cell injection (Figure 4H). In contrast, wild-type CD8 mice showed a specific decrease in fear conditioning responses to contextual learning at 6 months after T cell injection, and decreased responses to both contextual and cue learning at 11 months (Figure 4I). These results suggest that cognitive impairment in the wild-type CD8 group was initially limited to hippocampal function (necessary for contextual FC), but in the later, more advanced stages, it impaired both hippocampal and amygdala function (necessary for cue FC). A similar pattern of progressive cognitive impairment occurs in human Alzheimer's disease. Contextual learning performance at 6 months also correlated with brain volume, demonstrating its relationship to neurodegeneration.

[0089] To independently confirm cognitive deficits, spontaneous alternating behavior was measured 12 months after injection. This test is based on the mouse's preference to explore two paths alternately and requires memory of previously entered paths. The lowest possible score of 50% represents random selection of paths and reflects either a lack of short-term memory or a lack of preference. The SA of the control PBS group was 55–56%, comparable to the published wild-type value (27), while the SA of the wild-type CD8 group was 50% (Figure 4J). To test whether this reflected a deficit in memory or preference, the inventors administered the Burns maze test, a hippocampus-dependent definitive assessment of memory and learning, at 14 months. Wild-type CD8 nude mice showed no improvement in learning the maze over the first four-day training period, while all other groups showed considerable improvement (Figure 4K). Given this initial deficit, it was expected that wild-type CD8 mice would also have impairments during the memory retention and reverse learning phases of the maze (Figures 4L-4N). Similar to fear conditioning, a significant correlation was found between performance on the Burns maze and brain mass. Therefore, wild-type CD8 nude mice, while not exhibiting overt motor function deficits, showed progressive, severe, and persistent learning and memory impairments.

[0090] We further investigated whether performance on the Barnes Maze was associated with an increase in the Tau and / or Aβ evaluation indices. Low performance on the Barnes Maze (total waiting time below median = BM) was also investigated. lo ) did not show a significant association with increased soluble p-tau, but did show a significant association with increased tau PHF in Western blotting. In contrast, poor labyrinth performance was not significantly associated with either triton-soluble Aβ40 / Aβ42 or guanidine hydrochloride-soluble Aβ40 / Aβ42 according to ELISA. Thus, as reported in human Alzheimer's disease, tauopathy reflected cognitive impairment more than amyloidopathy in wild-type CD8 nude mice.

[0091] Cellular mechanisms of hiT cell-mediated neurodegenerative symptoms To determine the mechanisms involved in hiT cell-mediated neurodegenerative symptoms, CD8+ T cells derived from knockout donors lacking perforin 1 or IFNγ, key effectors of T cell lytic and pro-inflammatory activity, respectively, were injected into B6.Foxn1 mice. CD8+ T cells lacking either gene (Prf1 and Ifnγ, respectively) proliferated at a similar rate to wild-type cells in B6.Foxn1 recipients (Figure 1J), consistent with previous studies. Nevertheless, neither perforin 1-deficient nor Ifnγ-deficient CD8+ T cell recipients (PrfKO-CD8 group and IfnγKO-CD8 group, respectively) showed an increase in soluble Aβ or p-tau / PHF at any time point (Figures 3B and 3F). However, ifnγKO-CD8 mice showed only slightly reduced accumulation of amyloid plaques and silver-stained cells in the hippocampus and entorhinal cortex compared to the wild-type CD8 group, but these did not extend to the cingulate cortex (Figure 3D, Figure 3H). IfnγKO-CD8 mice also showed reduced astroglial and microglial proliferation (Figure 3I, Figure 3J), but unlike the PrfKO-CD8 group, CD8+ T cells were present in the brain in significant numbers 15 months after injection of these cells (Figure 3O, Figure 3P). The ifnγKO-CD8 group also showed reduced brain volume and NeuN at 15 months after injection. + The significant increase in both cells was unexpected. Finally, both the PrfKO-CD8 and IfnγKO-CD8 mice showed significantly reduced cognitive function at 11–15 months. Thus, the PrfKO-CD8 mice showed no evidence of pathophysiology on any criterion, including an increase in CD8+ T cells in the brain, while the IfnγKO-CD8 mice maintained some molecular pathophysiology, although they did not show evidence of neurodegeneration or cognitive decline.

[0092] CD8+T in age-related cognitive decline RM CD8+T RM It caused cognitive neurological symptoms in nude mice, but immune response mice It was unclear whether these cells also mediate age-related neuronal deficits in mice. CD103 increases in the brains of aged mice. RM This is a characteristic of T in the brain. RM This is important for regression. We demonstrate that CD103 gene deficiency primarily affects CD8+ T cells (Figure 5A) and reduces CD8 content in the brain (Figures 5B, 5C). Young and aged CD103-deficient mice performed similarly to wild-type counterparts during the training period of the Barnes maze, despite decreased walking activity with age (Figures 5D, 5E). In contrast, aged CD103-deficient mice performed slightly better during the memory retention and reverse learning phases (Figures 5F, 5G), and showed a significant reduction in the number of errors in the Barnes maze, the opposite of the age-related differences recorded in mice of this strain (Figure 5H). Thus, CD103 deficiency protected aged mice from age-related cognitive decline. CD103 deficiency primarily affects CD8+ T cells, and CD103 + Only the cells increase with age, either inside or outside the brain, thus contributing to the decline in cognitive function during aging. + CD8+T RM The involvement of CD8+T is confirmed. Age-related cognitive decline is a strong predictor of future neurodegenerative symptoms, and this confirms that CD8+T RM This is further associated with disease-related dementia (such as Alzheimer's disease).

[0093] hiT cell phenotype, effector proteins, and specificity in the brain of human Alzheimer's disease patients. To investigate the possible association of hiT cells with human Alzheimer's disease, we focused on perforin 1 and CD8 in the brains of patients with this disease, given that IFNγ is already known to be associated with disease risk. Western dysplasia revealed the expected antibody specificity (Prf1 at 68–75 kDa, CD8α at 33–35 kDa) and the expected punctate pattern of anti-Prf1 stained lymphocyte nuclei (Figure 6C). Western dysplasia signals for perforin 1 and CD8 correlated (n=6; r=0.8155, P=0.048), with both increasing in the cortex of patients with mild rather than severe Alzheimer's disease, and perforin 1 reaching statistical significance (Figure 6C). The perforin 1:CD8 signal ratio also significantly increased in the brains of patients with severe Alzheimer's disease, consistent with the long-term qualitative changes in lymphocyte lysis composition observed in hiT cell-carrying mice (Figure 6B). To further investigate this, we look at [APP], a T cell epitope that increases in hiT cell-carrying mice. (471-479) A pHLA-A2 multimer was created for a human T cell epitope similar to [ ]. Hippocampal sections from patients with severe Alzheimer's disease and normal-age patients containing anti-CD8 and this multimer or a control multimer were stained, and the proportion of epitope-reactive CD8+ T cells was quantified. After subtracting the staining of the negative control, APP (471-479) CD8+ T cell responsiveness to APP was significantly increased in the diseased population (Figure 6E, Figure 6F; P=0.002). While overall CD8+ T cell levels were not significantly elevated in the brains of patients with this disease, as was the case in hiT cell-carrying mice, they were slightly elevated (n=10; 1.6±0.29 vs. 2.3±0.55, P=0.31). Thus, APP-responsive CD8+ T cells were increased in the brains of patients with severe Alzheimer's disease, similar to the brains of hiT cell-carrying nude mice.

[0094] The table below lists the number of groups and the validation experiment. Validation: WB = Western blot, Morphology = Expected morphology obtained by tissue staining, WB (adsorption) = Expected positive signal by Western blot using negative antigen adsorption control, huAD = Additional expected morphology obtained in brain tissue from human AD patients, Co-staining = Staining with a second cell type-specific reagent (anti-CD8 antibody). [Table 1]

[0095] Nude mice possessing hiT cells showed several similarities to human neurodegenerative disorders, particularly Alzheimer's disease. Specifically, these mice exhibited neuroinflammation, silver-stained (fibrillary) neuronal inclusions, synaptic and neuronal loss accompanied by brain atrophy, and early Aβ accumulation and late amyloid plaque accumulation, similar to progressive cognitive impairment. Some of these features were not seen in mice that merely expressed familial Alzheimer's disease gene mutations. Most notably, these included neuronal loss accompanied by brain atrophy and neurofibrillary inclusions. However, there were differences between the neurological symptoms of Alzheimer's disease or FAD-type mouse models and those of hiT-cell-possessed nude mice. Clearly, only Aβ40 was increased, without the presence of Aβ42, and the amyloid plaques were overwhelmingly diffuse rather than mature. Nude mice also did not exhibit the acellular "ghost tangles" typically seen in human Alzheimer's disease.

[0096] Patients with the Iowa-type APP mutation primarily exhibit a pattern of increased Aβ40, a feature that distinguishes hiT cell-bearing nude mice from most human Alzheimer's disease models as well as most transgenic models. This may be partly due to the prominent vascular amyloid in hiT cell-bearing nude mice, where the predominantly Aβ40 composition may mask other Aβ species. Mouse Aβ40 exhibits a pattern opposite to that of human Aβ peptides, but also has a reduced efflux rate compared to Aβ42, which is expected to promote the retention of more Aβ40. Factors that specifically inhibit Aβ42 fiber assembly in the rodent brain may further contribute to the dominance of Aβ40. On the other hand, ghost tangles differ from other neurofibrillary structures in that they are primarily composed of 3R tau, which is virtually absent in adult mice. Therefore, the discrepancies between hiT-carrying nude mice and human Alzheimer's disease can be explained by a combination of technical and species-specific factors. Nevertheless, the unique symptoms of these mice revealed other similarities to the human disease. For example, cognitive impairment in hiT-carrying nude mice, which progressed from hippocampus-dependent tasks to amygdala-dependent tasks, correlated better with p-tau / PHF levels than with Aβ levels and correlated with brain atrophy in multiple behavioral tests. Against this background, it was not surprising that increases in both CD8 and perforin 1 were observed in the brains of early Alzheimer's disease, which was similar to the increase in effector-active CD8+ T cells in hiT-carrying nude mice. Furthermore, App in severe disease [471-479] The increased CD8+ T cell response to this is nearly identical to that of hiT-carrying nude mice (App [470-478] This was directly proportional to the proliferation of CD8+ T cells that respond to ).

[0097] The lytic and pro-inflammatory T-cell effector functions had separate effects on the neuropathological features of hiT-cell-carrying mice. Perforin 1 deficiency suppressed CD8+ T-cell retention in the brain, as well as all neuropathological and symptomatic features. In contrast, Ifnγ deficiency allowed the accumulation of amyloid and neurofibrillary structures, albeit with a limited distribution reminiscent of early preclinical Alzheimer's disease. This indicates that IFNγ promotes disease progression, as reported previously. Deficiencies in astroglial and microglial proliferation in IfnγKO-CD8 mice are also consistent with earlier studies showing that IFNγ promotes neuroinflammation in separate Alzheimer's disease models. However, unexpected increases in NeuN, drebrin, and brain volume in IfnγKO-CD8 mice suggest that IFNγ may also regulate neurodegeneration independently of neuroinflammation. Considering these different effects, perforin 1 + or IFNγ + It will be interesting to determine whether each of these CD8+ T cells can serve as a biomarker for the onset and progression of Alzheimer's disease.

[0098] The presence of HLA-DR risk alleles in Alzheimer's disease and Parkinson's disease suggests that T cells may play a significant role in the pathogenesis of neurodegeneration. Furthermore, recent discoveries of lymphatic vessels in the brain may provide a structural basis for the overall involvement of T cells in brain pathophysiology. While not all studies have reported an increase in CD8+ T cells or other features common to autoimmune diseases in Alzheimer's disease, the nature of T cell involvement remains largely unknown. It is reasonable to hypothesize that lytic autoreactivity may contribute to hiT cell-induced neurological symptoms, but these symptoms differed in several ways from the classical autoimmune neurodegeneration characteristic of multiple sclerosis or experimental autoimmune encephalomyelitis. For example, these symptoms were more dependent on CD8+ T cells than CD4+ T cells, were improved rather than worsened by IFNγ deficiency, and did not involve high-density immune infiltration. Additionally, hiT-carrying mice lacked the pronounced motor impairments of MS and EAE. This, along with the Alzheimer's disease-like neurological symptoms, strongly suggests that hiT cell-carrying nude mice do not exhibit MS-like autoimmune neurodegeneration.

[0099] hiT cell-mediated neurological symptoms, in conjunction with FAD mutations, may potentially produce a truly complete Alzheimer's disease-like pathological and symptomatic profile. It is equally important to investigate whether this complete disease-like feature in hiT cell-carrying mice is attainable in knock-in lines of the human Aβ (App) and / or tau (Mapt) genes, and to question the overall role of the lineage background in regulating neurological symptoms. Most importantly, confirming the association of hiT cells with sporadic Alzheimer's disease, its prodromal state, and risk factors is crucial, given the biomarker capabilities and pathogenesis of these cells. For now, our findings indicate that hiT cells overcome the mouse's normal resistance to the onset of age-related Alzheimer's disease-like neurodegeneration and the development of neurofibrillary inclusions. This represents the first solitary physiological factor and the first age-related immune cell feature to directly promote age-related Alzheimer's disease-like neurodegeneration. The findings described herein represent the first evidence that abnormal CD8+ T cells promote tissue degeneration in age-related disease states. It is thought that hiT cells, which react to different tissue antigens, may cause damage to the brain or other areas of the body. Therefore, this hiT model and age-related CD8+ T cell dysfunction may also be related to other age-related disorders, and possibly to the widespread tissue degeneration observed during aging itself.

[0100] Example 2: Standard operating procedure for HLA-peptide antigen multimer staining for flow cytometry principle This procedure describes a method used to determine the percentage of CD8+ T cells, T hybridoma cells, or cultured T cells that stain positively with MHC class I tumor antigen tetramers. Whole blood or PBMCs, T hybridoma cells, or cultured T cells derived from human subjects are divided into alicots and placed in U-bottom 96-well microtiter plates, followed by staining with monoclonal antibodies (mAbs). These mAbs, anti-CD8 and anti-KLRG1 or anti-CD103, recognize cell surface markers in subpopulations of aged T cells. Subsequently, cells are stained with pHLA multimers that recognize antigen-specific receptors on T cells. After incubation, these cells are washed to remove unbound reagents, and these cells are analyzed using flow cytometry. The percentage of PBMCs bound to tumor antigen tetramers is determined using the percentage of cells that enter the electronic gate defined by the control strain.

[0101] Specific requirements All reagents and instruments must be sterilized before use. Maintaining sterility during the procedure is not required but is recommended. Sterilized instruments must be brought in in their labeled manufacturer packaging and opened and used only within a sterile hood. Instruments may be sterilized in autoclavable sterilization pouches (Fisher catalog number 91015 as a suggested supplier). All processing must be performed within a laminar flow hood approved for handling human cells. Universal precautions for handling human tissues are required.

[0102] Materials / Reagents Pipettes (sterilized): P20 Eppendorf (VWR pipette, Calibrite INC service) and P200 Eppendorf (VWR pipette, Calibrite INC service) Pipette tips (sterilized, with filter): ART 20μl nuclease / pyrogen-free tips (Fisher, 2149P) and ART 200μl nuclease / pyrogen-free tips (Fisher, 2069) Sterile Dulbecco's phosphate-buffered saline (PBS) (Invitrogen, 14040-133) Sterilized FBS (Gemini Bio. Products, 100-106) Sterilized 2% FBS and 98% PBS solution Human anti-CD8 (Pharmagen, product number unknown, supplied by T-Neuro) Human anti-KLRG1 (Pharmagen, product number unknown, supplied by T-Neuro) Human anti-CD103 (Pharmagen, product number unknown, supplied by T-Neuro) Human HLA tumor antigen tetramers, pentamers, or dextramers (Her-2, Mart-1, gp100) supplied by T-Neuro. Coulter, ProImmune, Immudex, various numbers 4% paraformaldehyde (Sigma, 55F-0730) A solution of 70% ethanol and 30% distilled water Sterilized U-shaped bottom 96-well microtiter plate (Costar, 3595) Sterilized FACS tubes Container with lid for crushed ice A clean 1-liter container for bleach Bleach

[0103] Equipment A refrigerated centrifuge capable of rotating at 1400g, with at least two microtiter plate adapters. FACScan II or other flow cytometer (minimum 3 colors) (Becton Dickinson, Cytomation, etc.) Note: If you are not in contact with the CRO, schedule the FACS operation in advance with the facility operator. Quality control (internal): Record results in appropriate laboratory logs and notebooks. Include observations and, if GLP is followed, a signature from a witness who did not perform the procedure. The procedure form and checklist must be completely filled out and signed by the head of the laboratory (quality assurance officer) on the same day. Obtain a printed copy of the FACScan flow cytometer settings for the FACS operation day from the FACS operator. Alternatively, obtain the .fcs file from the person in charge regarding the processing and analysis.

[0104] method FACS staining of cell surface markers Using a P200 pipette wiped with 1.70% ethanol, add 2.5 × 10¹⁶ tetramers in PBS / 2% FBS to each of four wells of a U-bottom 96-well microtiter plate, up to a maximum of 2.5 × 10¹⁶ tetramers per well. 5 Cells (maximum 1 x 10) 6 Transfer PBMCs or whole blood (e.g., 2 tetramers = 4 wells; if you are only planning on anti-KLRG1 antibody staining, this will be exactly 2 wells per sample). Note: The following procedure does not need to be performed inside the clinical laboratory or laminar flow hood. 2. Using the centrifuge located in clinical laboratory D2095, centrifuge the plate at 1400 rpm for 5 minutes. 3. Discard the contents of the bleach into a clean plastic waste container containing approximately 250-500 ml of bleach, then discard the supernatant. Afterwards, place the plate on a clean paper towel to dry. 4. Transfer the 96-well plate containing the cells and antibodies to an ice container filled with ice. Using a clean P200 wiped with 5.70% ethanol and a sterile pipette tip, resuspend the cells in the wells with 50 μl of the following antibody cocktail. a) 5 μl anti-CD8 APC +5 μl of anti-CD8103 FITC +35 μl of PBS / 2% FBS; or replace with antibody for another marker. b) 5 μl of anti-CD8 APC +5 μl anti-KLRG1 FITC +35 μl PBS / 2 %FBS Each HLA tumor antigen tetramer to be analyzed should contain the same two cocktails (e.g., 2 tetramers + up to 2 strains = a total of up to 4 wells). Incubate the cells on ice in the dark for 30 minutes (cover the U-bottom microtiter plate and label it so that no one can tip it over). Note: Use a clean pipette tip for each administration of a different antibody cocktail. Using a clean P200 pipette and a sterile pipette tip, wiped with 6.70% ethanol, add 200 μl of PBS / 2% FBS to each well. Use a clean tip for each well to avoid contamination. 7. Set the brake to high and centrifuge the plates at 1400 rpm for 5 minutes. 8. Discard the contents of the bleach into a clean plastic waste container containing approximately 250-500 ml of bleach, then discard the supernatant. Afterwards, place the plate on a clean paper towel to dry. Using a clean P200 wiped with 9.70% ethanol and a sterile pipette tip, the pre-incubated wells are resuspended with each of the three antibody cocktails and each HLA tumor antigen tetramer solution (one well per tetramer to be analyzed; 2 wells for 2 tetramers total) as follows: c) 5 μl HLA-APP-PE + 35 μl PBS / 2% FBS = 1 well d) 5 μl HLA-empty PE + 35 μl PBS / 2% FBS = 1 well 10. Incubate the above cells in the dark at room temperature (exactly 25°C) for 30 minutes (cover the plate and label it so that no one can tip it over). Using a clean P200 wiped with 11.70% ethanol and a sterile tip, Add 200 μl of ice-cold PBS / 2% FBS to each well. Use a clean tip for each well to avoid contamination. 12. Using the centrifuge located in the clinical laboratory (D2095), set the brake to high and centrifuge the 96-well plate at 1400 rpm for 5 minutes. 13. Discard the contents of the bleach by flicking it into a clean plastic waste container containing approximately 250-500 ml of bleach. Then, place the plate on a clean paper towel to dry. Using a clean P200 pipette tip wiped with 14.70% ethanol and a sterile pipette tip, resuspend the cells in each well with 150 μl of ice-cold PBS / 2% FBS. Resuspend by polishing. Use a clean tip for each well to avoid contamination. Using a clean P200 wiped with 15.70% ethanol and a sterile pipette tip, transfer the above cells to an analytical FACS tube. 16. Selection process for delaying cell analysis (Note: This process is directed only at stained PBMCs, not whole blood.) 16.1. Using a clean P200 pipette wiped with 70% ethanol and a sterile pipette tip, fix the cells in the 96-well plate with 50 μl of 4% paraformaldehyde. Use a different tip for each sample and ensure that the cells and formaldehyde are thoroughly mixed by pipetting up and down within the tube. 17. Take the tube along with the FACS analysis request form to room D4029 on the 4th floor. 18. Obtain the results, save them in the laboratory notebook assigned for FACS analysis, and download the raw.fcs file for subsequent analysis. Report of results 1. Within 24 hours of receiving the FACS results from the operator, all data will be reviewed and approved by the colleague and principal investigator responsible for performing this assay. 2. The analyzed data originates from a minimum of 50,000 collection events (more than 250,000 is preferable) and is presented as the percentage of cells within the viable lymphocyte gate determined by forward-right scatter and side-right scatter. 3. Minimal staining must be achieved for both pHLA-empty cells (more than 0.7% of cells in the lymphocyte gate) and anti-CD8 cells (more than 3.0% of cells in the lymphocyte gate) to qualify for subsequent analysis. 4. The procedure form must be completely filled out (including the check for registration of equipment usage records) and signed by the head of the laboratory on the same day. Technical Notes 1. Use only sterile reagents and equipment. 2. Work only inside the designated hood. Turn on the hood 10 minutes before use. 3. Before use, wipe the hood clean with 70% ethanol. 4. Do not return a pipette used in a flask back into the reagent. 5. Change your gloves every time you take your hands out of the hood. 6. Always wear a lab coat, gloves, and protective sleeve when working with any blood components. 7. Cells can be stored at 4°C for up to one week before analysis by flow cytometry.

[0105] Various embodiments of the present invention have been described in the context of modes for carrying out the invention. These descriptions directly illustrate the above embodiments, and it will be understood that those skilled in the art will be able to conceive of modifications and / or variations to these particular embodiments presented and described herein. Any such modifications or variations that fall within the scope of this description shall be similarly included in these embodiments. Unless otherwise noted, the inventors intend that the words and phrases in this specification and in the claims are to be used in the ordinary and common sense to those skilled in the art.

[0106] The above description of various embodiments of the invention known to the applicant at the time of filing this application is provided for illustrative and explanatory purposes only. This description is not intended to be exhaustive, nor is it intended to limit the invention to the precisely disclosed form, and numerous modifications and variations are possible in light of the above teachings. The described embodiments are useful in illustrating the principles of the invention and their practical applications, and also enable those skilled in the art to utilize the invention in various embodiments and with various modifications to suit specific conceivable uses. Accordingly, the invention is not limited to the specific embodiments disclosed for carrying out the invention.

[0107] While specific embodiments of the present invention have been presented and described herein, modifications and alterations can be made based on the teachings herein without departing from the present invention and its broader aspects, and it will be apparent to those skilled in the art that the appended claims are encompassed within that scope and that all such modifications and alterations are in the true spirit and scope of the present invention. In general, it will be understood to those skilled in the art that the terms used herein are generally "open" terms (for example, the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "at least having," and the term "includes" should be interpreted as "including but not limited to," etc.).

[0108] Where used herein, the terms “comprising” or “comprises” refer to compositions, methods, and their individual components that are useful for the embodiments, yet are open to including elements that are useful or not. Generally, the terms used herein are generally “open-ended” terms (for example, “including” should be interpreted as “including but not limited,” “having” as “at least having,” and “includes” as “including but not limited,” etc.). While the open-ended term “comprising,” as a synonym for terms such as “including,” “containing,” or “having,” is used herein to describe and claim the present invention, the present invention or embodiments thereof may also be described using alternative terms such as “consisting of” or “consisting essentially of.” <Note> The embodiments of the present invention include the following: <Section 1> A method for treating, suppressing, reducing the severity of, or promoting the prevention of age-related cognitive decline, mild cognitive impairment, pathological neurodegeneration, or combinations thereof in a target population, The method comprising administering a therapeutically effective dose of one or more of the following to the subject: a differentiation antigen group (CD103) inhibitor, a perforin 1 inhibitor, and an interferon-gamma (IFNγ) inhibitor. <Section 2> The CD103 inhibitor is administered, The method according to claim 1, wherein the CD103 inhibitor is an anti-CD103 antibody and comprises a PE anti-human CD103 antibody derived from clone Ber-ACT8, a mouse anti-human CD103 monoclonal antibody (mAb) derived from clone 2G5.1, a humanized antibody of 2G5.1, OX-62, a humanized antibody of OX-62, an anti-mouse CD103 monoclonal antibody derived from clone 2E7, a humanized antibody of 2E7, or paxillin. <Section 3> The perforin 1 inhibitor is administered, The method according to claim 1, wherein the perforin 1 inhibitor comprises diarylthiophene or GSK2126458. <Section 4> The aforementioned IFNγ inhibitor is administered, The method according to claim 1, wherein the IFNγ inhibitor comprises mesopram or locaglamide. <Section 5> The method according to claim 1, wherein the perforin 1 inhibitor and the IFNγ inhibitor are administered. <Section 6> The method according to claim 1, wherein the CD103 inhibitor, the perforin 1 inhibitor, and the IFNγ inhibitor are administered. <Section 7> The aforementioned one or more inhibitors affect CD8+ resident memory T cells (T RM ) or the aforementioned details It regulates effector CD8+ T cells derived from cells, and the CD8+ T RM or the above The method according to item 1, wherein effector CD8+ T cells inhibit the binding or reaction to amyloid precursor protein (APP) peptide. <Section 8> The method according to claim 7, wherein the binding to or inhibition of the reaction with APP peptide includes the binding to or inhibition of the reaction with APP peptide of SEQ ID NO: 8 in the brain. <Section 9> The method according to item 1, wherein the subject is a person aged 50 or older, 55 or older, 60 or older, 65 or older, or 70 or older. <Section 10> Compared to before administration of one or more of the aforementioned inhibitors, or compared to control subjects who were not administered one or more of the aforementioned inhibitors, the activity of the effector CD8+ T cells is reduced, and / or the CD8+ T RM The method described in item 7, in which migration from the peripheral system to the brain is reduced. <Section 11> The method according to claim 1, wherein the CD103 inhibitor, the perforin 1 inhibitor, and the interferon-γ (IFNγ) inhibitor independently comprise an antibody, an antigen-binding fragment of an antibody, a small molecule, or a nucleic acid. <Section 12> The method according to claim 1, wherein the pathological neurodegeneration includes one or more of multiple sclerosis, Parkinson's disease, and Alzheimer's disease. <Section 13> The method according to claim 1, wherein the age-related cognitive decline or mild cognitive impairment has one or more symptoms among loss of short-term or long-term memory, decreased ability to maintain concentration, and decreased ability to solve problems. <Section 14> The subject is a human subject, Further comprising identifying the human subjects who are prone to pathological neurodegeneration or who have pathological neurodegeneration prior to the administration of the said administration, CD103+ commensal memory CD8+ T cells (CD8+T) in human blood RM This includes detecting an increase in the presence of ) compared to values ​​obtained from the same human subjects at a younger age without symptoms of age-related cognitive decline, or compared to values ​​obtained from one healthy human subject or a pool of multiple healthy human subjects without symptoms of age-related cognitive decline, The aforementioned pathological neurodegeneration includes Parkinson's disease, multiple sclerosis, or Alzheimer's disease. The method according to item 1, wherein the age-related cognitive decline has one or more symptoms among loss of short-term or long-term memory, decreased ability to maintain concentration, and decreased ability to solve problems. <Section 15> The aforementioned detection step is CD103+CD8+T RMThe method according to item 14, further comprising detecting an increase in CD8A and CD44 levels in the following location. <Section 16> The method according to item 14, wherein the human subject is 65 years of age or older. <Section 17> A method for treating, suppressing, reducing the severity of, or promoting the prevention of age-related cognitive decline or pathological neurodegeneration, including multiple sclerosis, Parkinson's disease, or Alzheimer's disease, in a target population, This includes administering a therapeutically effective dose of the vaccine to the subject, The vaccine contains an amyloid precursor protein (APP) peptide selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 2, and combinations thereof, or APP. The aforementioned method. <Section 18> The method according to item 17, wherein the subject is a person aged 40 or older, 50 or older, 60 or older, or 70 or older. <Section 19> A method for identifying human subjects who are prone to age-related cognitive decline or pathological neurodegeneration, or who are already experiencing age-related cognitive decline or pathological neurodegeneration, CD103+ resident memory CD8+ T cells (CD8+T) in blood samples obtained from human subjects exhibiting one or more of the following symptoms: loss of short-term or long-term memory, decreased ability to maintain concentration, and decreased ability to solve problems. RM This includes detecting an increase in the presence of ) The aforementioned method. <Section 20> The aforementioned CD103+CD8+T RM The increased presence of any one of the above one or more symptoms The method described in paragraph 19, which is compared to values ​​obtained from a single healthy human subject or a pool of multiple healthy human subjects.

Claims

1. A pharmaceutical composition for treating, suppressing, reducing the severity of, or promoting the prevention of age-related cognitive decline, mild cognitive impairment, pathological neurodegeneration, or combinations thereof in a target population, It contains one or more therapeutically effective doses of differentiation antigen group (CD103) inhibitors, perforin 1 inhibitors, and interferon-gamma (IFNγ) inhibitors. The CD103 inhibitor is an anti-CD103 antibody, and is selected from the group consisting of PE anti-human CD103 antibody derived from clone Ber-ACT8, mouse anti-human CD103 monoclonal antibody (mAb) derived from clone 2G5.1, humanized antibody of 2G5.1, OX-62, humanized antibody of OX-62, anti-mouse CD103 mAb derived from clone 2E7, humanized antibody of 2E7, and paxilin. The perforin 1 inhibitor is selected from the group consisting of diarylthiophene and GSK2126458. The IFNγ inhibitor is selected from the group consisting of mesopram and locaglamide. Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, comprising the perforin 1 inhibitor and the IFNγ inhibitor.

3. The pharmaceutical composition according to claim 1, comprising the CD103 inhibitor, the perforin 1 inhibitor, and the IFNγ inhibitor.

4. The pharmaceutical composition according to claim 1, wherein the subject is a human being aged 50, 55, 60, 65, or 70 years or older.

5. The pharmaceutical composition according to claim 1, wherein the pathological neurodegeneration includes one or more of multiple sclerosis, Parkinson's disease, and Alzheimer's disease.

6. The pharmaceutical composition according to claim 1, wherein the age-related cognitive decline or mild cognitive impairment has one or more symptoms among loss of short-term or long-term memory, decreased ability to maintain concentration, and decreased ability to solve problems.

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