Methods and systems for identifying and treating pathological neurodegeneration and age-related cognitive decline

Abstract

The present invention provides a composition for the diagnosis, prevention, and / or treatment of age-related cognitive decline, including pathological neurodegeneration, as well as diagnostic, preventive, and / or therapeutic methods using the composition. [Solution] Provided are methods and systems for diagnosing, preventing, and treating one or both of age-related neurodegeneration and cognitive disorders. Provided are methods for protecting at-risk elderly subjects, subjects with mild cognitive impairment, and / or subjects with pathological neurodegeneration from cognitive decline and / or reducing the severity of such decline, comprising administering a CD103 inhibitor, which is an inhibitor of an effector molecule of CD8+ resident memory T cells, and / or a tolerogenic vaccine. Provided are methods for identifying subjects susceptible to or experiencing age-related neurodegeneration, comprising detecting elevated levels of CD103+ resident memory T cells. Provided are kits for collecting and quantifying CD103+CD8+ resident memory T cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 630,129, filed February 13, 2018, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to the diagnosis, prevention and treatment of age-related or pathological neurodegeneration. [Background technology]

[0003] Aging contributes to the onset and / or progression of multiple diseases, but the specific ways in which aging influences the dynamics of individual diseases remain largely mysterious. Chronic inflammation is increasingly recognized as an important contributor to age-related diseases, including cancer, cardiovascular disease, cancer, and neurological conditions such as stroke, trauma, and neurodegeneration.

[0004] T cells are master regulators of inflammation throughout the body, and their misregulation promotes chronic inflammation. CD8+ T cells can acquire the capacity for self-destruction, particularly when the peripheral T cell pool is depleted and these cells undergo homeostatic proliferation. Such depletion can occur gradually during aging due to thymic involution while generating new T cells, or more rapidly due to stress, trauma, or infection. Age-related T cell proliferation continues into late middle age in humans and occurs ubiquitously, making it difficult to study whether pathological factors are responsible for aberrant T cell proliferation instead of the natural aging process. Furthermore, aberrant age-related T cell proliferation is relatively rare in experimental rodents, and even if it does occur, its functional consequences are often offset by persistent thymic activity during aging.

[0005] Introduction of T cells into lymphopenic hosts rapidly leads to homeostatic proliferation in those hosts, and the resulting cells can promote artificial autoimmunity in experimental rodents. Nevertheless, the relationship between this more rapid proliferation and age-related T cell abnormalities is less clear, and in most cases, no recognizable age-related diseases are known to be driven by this relationship.

[0006] While aberrant CD8+ T cell clones expand specifically in most aging humans, this process is counterbalanced by compensatory processes in aging mice. Changes in memory CD8+ T cells are also among the distinct physiological differences observed between mouse models and humans with Alzheimer's disease; these cells decrease in mouse models and increase in humans with Alzheimer's disease. Recent studies have convincingly demonstrated that memory CD8+ T cells are increased in the circulatory system and / or central nervous system (CNS) of Alzheimer's disease patients. While these increases are typically accompanied by significant changes in other T cells, they tend to correlate more strongly with tauopathy and / or cognitive decline. Therefore, age-related homeostatic proliferation and the resulting aberrant memory CD8+ T cells may be a novel physiological factor in mice that may influence resistance to the full spectrum of Alzheimer's disease pathology. However, this remains difficult to address due to the widespread presence of age-related CD8+ T cells in healthy humans, their rarity in mice, and their occurrence alongside other hallmarks of ageing in all species.

[0007] Accordingly, 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, as well as diagnostic, preventive, and / or therapeutic methods using the composition.

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

[0009] All publications within this specification are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description contains information that may be useful in understanding the present invention. This is not an admission that any of the information provided herein is prior art or relevant to the inventions claimed in this application, or that any of the publications specifically or expressly referenced are prior art. Summary of the Invention

[0010] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods, which are meant to be representative and exemplary and are not meant to limit the scope of the invention.

[0011] and administering to at-risk elderly subjects, subjects with mild cognitive impairment, and / or subjects with pathological neurodegeneration, a CD103 inhibitor, a CD8+ T RM In various embodiments, methods are provided that include administering an effector molecule inhibitor of CD8+ T (resident memory T cells), and / or a tolerogenic vaccine. RM The CD8+ T cells may be reactive or specific to amyloid precursor protein (APP) or an APP peptide. According to one aspect of the method, administering a vaccine comprising a CD103 inhibitor, a perforin 1 inhibitor, an interferon gamma (IFNγ) inhibitor, and / or an APP peptide results in increased CD8+ T cell response to APP or an APP peptide compared to before administration of one or more of the inhibitors or one vaccine, or compared to a control subject not administered one or more of the inhibitors. RM It is possible to inhibit the binding or reaction of

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

[0013] A method for identifying a subject susceptible to or suffering from age-related neurodegeneration, including Alzheimer's disease, comprising detecting CD103-positive resident memory T cells (T RM The method may include detecting an elevated level of

[0014] Before, during, and / or between treatments for memory impairment, the subject has CD103-positive CD8+ T cells. RM A kit is provided that includes a sample collection device for collecting and quantifying the .alpha., and optionally an operating manual.

[0015] A system for identifying and / or screening candidate therapeutic, preventive, and / or diagnostic agents for human cognitive decline or age-related neurodegeneration, comprising: a CD44 antibody derived from a rodent (e.g., a mouse); hi CD123 + CD127 hi KLRG1 + CD103 + The above system is provided to include a resident memory CD8+ T cell phenotype. In some embodiments, this CD44 hi CD123 + CD127 hi KLRG1 + CD103 + A resident memory CD8+ T cell phenotype can be obtained by administering resident memory CD8+ T cells to athymic mice.

[0016] A method for identifying and / or screening candidate agents for treating or preventing age-related neurodegeneration in humans comprises: detecting the candidate agent in vitro in a manner similar to that described above for CD44 hi CD123 + CD127 hi KLRG1 + CD103 + by contact with resident memory CD8+ T cells or by contact with the CD44 hi CD123 + CD127 hi KLRG1 + CD103 + This may include administering the candidate agent to a model animal containing resident memory CD8+ T cells and determining a reduced level of CD103-positive resident memory CD8+ T cells, a reduced level of effector molecules on those cells, or a reduced migration of CD8+ T cells from the periphery to the brain of the animal.

[0017] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.

[0018] Reference is made to the drawings, which illustrate exemplary embodiments. The embodiments and drawings disclosed herein are to be considered illustrative rather than restrictive. [Brief explanation of the drawings]

[0019] [Figure 1A]Figures 1A-1G show that hiT cells exhibit an age-associated resident memory phenotype (hiTRM). Figures 1A-1D show results from spleen specimens, and Figures 1E-1G show results from brain analyses. Figure 1A shows representative flow cytometry analysis of age-related markers on splenic CD8+ T cells from young (<10 weeks) and old (>12 months) C57BL / 6 (B6) mice (labeled "young B6" and "old B6," respectively), and from young (6 weeks) B6.Foxn1 recipients (labeled "CD8→B6.Foxn1") 3-5 weeks after intravenous infusion of CD8+ T cells. Figure 1B shows the percentage of lymphocytes derived from flow cytometry and the mean fluorescence intensity (Figures 1C and 1D) from ≥6 mice per group. The proportion of mice with "diverse" TCR V β-chain D→J gene segment usage (more than three segments per brain) and specific D→J segments in the brains of young (<10 weeks, denoted "young B6"), middle-aged (6 months, denoted "middle B6"), and old (>12 months, denoted "old B6") B6 mice shows an age-dependent pattern of progressively decreasing diversity and increasing specific D→J segment usage (i.e., clonality; Figures 1E and 1F; columns appear in the defined order from left to right at the bottom of each panel). D→J diversity and segment usage were significantly correlated only between old B6 and young CD8→B6.Foxn1 brains (Figure 1G). *P<0.05, **P<0.01, ***P<0.005. Flow cytometry markers were analyzed by two-tailed t-test with at least five mice per group in at least three independent experiments. PCR compilation was performed using Pearson correlation 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 donor cell proliferation in amyloid precursor protein (APP)-deficient B6.Foxn1 mice. Purified CD8+ T cells from female C57BL / 6 or congenic knockout hosts were infused into 8-10-week-old female B6.Foxn1 recipients, B6.Foxn1-AppKO recipients, or B6.CD45.1 congenic recipients (Figure 1H). Blood was analyzed by flow cytometry 3 days later using the gate settings and antibodies against T cell markers as indicated (Figure 1I). The percentage of CD3ε+CD8+ cells among cells within the gate was summarized (Figure 1J). B6.Foxn1 mice were crossed with B6.App-knockout mice, and homozygous double mutants (B6.Foxn1-AppKO) were confirmed by PCR and phenotyping at Jackson Laboratories (Bar Harbor, ME). CD8+ T cell proliferation was assessed by CFSE dilution in B6.Foxn1 and B6.Foxn1-AppKO female recipients (Figure 1K; n = 3 B6.Foxn1 and n = 5 B6.Foxn1-AppKO; *P < 0.04, ***P < 0.00001 by two-tailed t-test on three independent experiments; n ≥ 5 mice / group on three or more independent experiments for all markers). [Figure 1I] Same as above [Figure 1J] Same as above [Figure 1K] Same as above [Figure 2A]Figures 2A-2E show that hiTRM responds to autoantigens and selectively invades the brain (i.e., brain CD8+ T cell phenotype after transfer into nude mice). Light scatter plots and gating of brain lymphocytes and CD8+ T cells in B6.Foxn1 recipients (Figure 2A). The percentage and phenotype of CFSE+ CD8+ T cells within brain lymphocytes in B6.Foxn1 recipients 3 days (Figure 2B) and 10 weeks (Figure 2C) after transfer. Figure 2D and 2E show enhanced staining using pMHC I multimers (custom dextramers synthesized by Immudex USA, Fairfax, VA) for the Trp-2-DCT(180-188) / H-2Kb epitope and the APP(470-478) / H-2Db epitope on KLRG1+CD8+ T cells in the brain (Figure 2D and 2E) and spleen (Figure 2E) of B6.Foxn1 recipients 10 weeks after infusion (*P<0.05 by two-tailed t-test for three or more independent experiments; 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-J show that induction of hiTRM increases CD8 and amyloid precursor protein (APP) / Ab expression in the brain (i.e., PCR and Western analysis of T cell and amyloid markers). PCR of the TCR Vβ chain D1→J1 gene segment (Figure 2F) and D2→J2 gene segment (Figure 2G) demonstrated a diverse T cell repertoire in young and aged C57BL / 6 (B6) mice, but limited TCR Vβ chain diversity in B6.Foxn1 recipients (CD8→B6.Foxn1) after 10 weeks of CD8+ T cells. B6.Foxn1 mice without prior infusion of wild-type CD8+ T cells lacked rearranged TCR products in the brain (i.e., visually confirmed to contain only germline "G" TCR products) (Figures 2F and 2G; note: segment J2.6 is a pseudogene). Western blots for CD8α (antibody clone 2.43; Figure 2H) and β-amyloid (antibody clone 4G8; Figure 2I) were performed in the hippocampus of excised brains from young (<5 months) C57BL6 (B6) mice and B6.Foxn1 hosts with and without adoptive transfer of CD8+ T cells from a young (6-8 weeks) B6 donor. CD8 protein is detectable at very low levels in B6 mice, but is undetectable in B6.Foxn1 mice unless wild-type CD8+ T cells were infused 10 weeks prior. "Ref" = spleen DNA or cell lysate from a 6-10 week-old female C57BL / 6 mouse used in the same analysis. Figure 2J shows the timeline of the study. [Figure 2G] Same as above [Figure 2H] Same as above [Figure 2I] Same as above [Figure 2J] Same as above [Figure 3A]Figures 3A-J show Aβ plaques and neurofibrillary disease symptoms in nude mice bearing hiT cells. Western swabs of detergent-soluble APP cleavage products (APPCl) in excised cortex and hippocampus 3 weeks after control or cell infusion (→) in the indicated recipients (Figure 3A). Cell / control recipients in Figures 3B-J are exclusively B6.Foxn1, and are at 15 months postinfusion unless otherwise indicated. Forebrain ELISA of the Triton-soluble fraction Aβ1-40 / 42 (Figure 3B). Parenchymal plaques with and without p-tau or curcumin counterstain in the indicated mouse groups (Figure 3C), and a summary of 4G8 area fraction in the entorhinal (Ent) cortex, cingulate (Cng) cortex, and hippocampus (Hippo) (Figure 3D). Figure 3E shows a Western forebrain analysis of detergent-soluble phospho-tau (p-tau) and paired helical filaments (PHFs) and a summary of signal quantification (Figure 3F). Figure 3G shows a brain with an inset of sequential p-tau → Gallias staining and silver-stained cells in an 18-month-old ADtg (Tg2576) mouse. Figure 3H shows a summary of the percentage of Gallias+ neurons, astrocytes (Gfap+), and microglia (Iba-1+; Figures 3I and 3J). For all analyses, *P<0.05, **P<0.01, and ***P<0.005 compared to the PBS group by two-tailed t-test with at least three independent experiments. [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 show that hiTRM induces fibrillar inclusions in brain cells at 6 months (i.e., separate staining for curcumin and thioflavin S in the dentate gyrus of nude mice bearing hiT cells). Hippocampal sections from the indicated groups (all B6.Foxn1 recipients except for AD-Tg = Tg2576 mice) were stained for 4G8 (Aβ) and curcumin 6 months after intravenous control / cell injection or at 14 months of age for AD-Tg (Figure 3K). Right-pointing arrows highlight Aβ deposits without curcumin co-staining. Upward-pointing arrows indicate colocalized Aβ and curcumin deposits, representing mature senile plaques. Downward-pointing arrows highlight curcumin+ structures without Aβ co-staining, i.e., non-amyloidotic fibrillar deposits. DAPI was not used in these stainings. The blue channel background is presented for anatomical reasons only. Figure 3L shows follow-up thioflavin S staining of the dentate gyrus of control / cell-injected B6.Foxn1 hiT recipients (PBS and wild-type CD8) and 20-month-old AD-transgenic (Tg) rats 6 months after cell injection. The brains of AD-Tg rats were used because they clearly contain tau PHFs (Cohen et al., 2013), but our technique did not allow for thioflavin S staining. [Figure 3L] Same as above [Figure 3M] Figures 3M and 3N show silver-stained neural structures in the experimental groups. Gallias silver staining of cortical and hippocampal brain regions, showing typical neurofibrillary tangle (NFT) morphology (inset), is shown 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 observed (inset). Individual images were from separate mice in each group (Figure 3M). Gallias structures in the hippocampus (left) and cortex (ctx, right) of nude mice bearing hiT cells (wild-type CD8) are compared with those in the cortex of a mouse with severe human AD (Braak stage VI) (Figure 3N). The magnification and scale are the same (20x) for all images in Figures 3M and 3N, and are also the same for insets. [Figure 3N] Same as above [Figure 3O] Figures 3O-3S show brain CD8+ T cells in hiTRM recipient B6.Foxn1 mice. Brains were co-stained for CD8 and p-tau (inset) in hippocampal and cortical brain sections from B6.Foxn1 recipients 15 months after infusion of wild-type, IFNγKO, or PrfKO CD8+ T cells or PBS (Figure 3O) and quantified. CD8+ cells were mostly isolated but occasionally interacted with p-tau+ neurons, as seen in Figure 3O (inset). Data for the groups are summarized in Figure 3P. The area of ​​significantly (**P<0.01, *P<0.05; two-tailed t-test compared with PBS control) altered astrocytes (GFAP, Figure 3Q), microglia (Iba-1, Figure 3R), or CD8+ T cells (CD8, Figure 3S) in Figures 3A-J or 3O-S correlated with the percentage of 4G8+ plaque area in each group. P values ​​for linear regression and Pearson correlation (r) are shown (Figures 3Q-S). [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 indexes and cognitive function in nude mice bearing hiT cells (i.e., hiTRM recipient B6.Foxn1 mice). Cell / control recipients in all panels are exclusively B6.Foxn1. NeuN and GFAP staining (Figures 4A and 4B) and cell counts in the hippocampus 15 months after cell / control injection (Figure 4C). Brain atrophy over time in the PBS and wild-type CD8 mice (amounts normalized to PBS control at each time point; Figure 4D). Representative forebrain Western (Figure 4E) and Western signals for NeuN, drebrin, and synaptophysin normalized to GAPDH (Figure 4F). Correlation of NeuN with brain volume (Figure 4G). Representative open field test at 13 months (Figure 4H). Figure 4I shows performance over time in the fear conditioning test, and Figure 4J shows spontaneous alternation (SA) at 12 months (Fig. 4J). Figures show Barnes maze learning (Fig. 4K; P by two-way ANOVA), retention (Fig. 4L), and reversal learning (Fig. 4M and Fig. 4N) at 14 months (black symbols = P compared to PBS and wild-type CD8, respectively). ***P<0.005, **P<0.01, *P<0.05, +P<0.1 by two-tailed t-test 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 show that CD103 deficiency primarily affects CD8+ T cells and their brain localization. Flow cytometry (Figure 5A), Western blot (Figure 5B), and Western blot (Figure 5C) data from CD103-deficient (B6.CD103KO) mice and age-matched wild-type (B6) mice (n = 8) are shown. The figures also show the results of open field tests (Figure 5D; columns appear in a defined order from top to bottom on the right side of the figure). CD103 deficiency primarily affected CD8+ T cells (Figure 5A), specifically reduced CD8 T cells in the brain (Figures 5B and 5C; in the absence of CD103 deficiency, CD8+ TRMs increased significantly in the aging brain), and slightly slowed locomotion with age (Figure 5D). [Figure 5B] Same as above [Figure 5C] Same as above [Figure 5D] Same as above [Figure 5E] Figures 5E-5H show that CD103 deficiency protects against age-related cognitive decline. The Barnes maze performance of young and aged CD103-deficient mice and wild-type mice is shown in Figures 5E-5H. These figures show the latency of the training period (Figure 5E), the latency of the memory retention period (Figure 5F), the latency of the reversal learning period (Figure 5G), and the number of entry errors in each period (Figure 5H). It has been reported that the primary age-related impairment in this strain in this test is entry errors. [Figure 5F] Same as above [Figure 5G] Same as above [Figure 5H] Same as above [Figure 6A]Figures 6A-6F show elevated hiT cell-associated metrics in human Alzheimer's disease brains. Figure 6A shows GFAP expression units in brains of subjects without Alzheimer's disease (no AD) compared to those in brains of subjects with AD. Figure 6B shows the percent change in gene expression levels compared to the corresponding GFAP expression levels in the subject brains. Figure 6C shows PRF1 Western and immunofluorescence, and quantification in age-matched normal (n = 6), mild (n = 5), or severe (n = 12) Alzheimer's disease brains (Figure 6D). Figure 6E shows Alzheimer's disease brains co-stained with anti-CD8 (Serotec) and APP(471-479) / HLA-A2 multimer (Immudex USA, Fairfax, VA), and quantification of epitope-reactive T cells (P = 0.002, two-tailed t-test) (Figure 6F). Overall levels of CD8+ T cells were unchanged (1.63±0.29 vs. 2.29±0.55 cells / vessel in Alzheimer's disease vs. normal aging controls; P=0.31, two-tailed 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 abnormal, APP-specific CD8+ TRM levels in patients at risk for age-related or disease-related cognitive decline (MoCA = 26-30) and in 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 reactive) (Figure 7B). [Figure 7B] Same as above [Figure 8] FIG. 8 shows a general model for CD8+ TRM-mediated brain effects. [Figure 9A]Figures 9A-9C show CD8+ TRM gene expression in human patient blood (Figures 9A and 9B) and hiTRM staining index in human patient blood (by flow cytometry) (in Figure 9C, 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 age-related 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 key genes, CD103, CD8A, and CD44, are significantly elevated in Alzheimer's disease, respectively. Patients with lower-than-average expression of the common T cell gene CD3D (Figure 10D) were excluded from the biomarker analysis to ensure that the predictive power was T cell-dependent. Patients younger than 65 years of age (Figure 10E) were also excluded to screen out rare early-onset Alzheimer's disease (AD) with alternative 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 true-positive and false-positive prediction rates for Alzheimer's disease (AD) using a three-gene panel (CD8, CD44, and CD103). After exclusion (described in the previous paragraph), 40 normal and 49 AD specimens remained for biomarker analysis in the high T cell group (Figure 11A), and 40 and 39 AD specimens 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 specimens, confirms that abnormal aging T cells are associated with AD and serve as a potent blood biomarker for late-onset disease. Including CD8A and CD44 along with CD103 in the receiver operating characteristic (ROC) analysis identifies the CD8+ TRM subpopulation more specifically than CD103 alone. Compared to the respective ROC curves based on CD103 alone shown in Figures 12A and 12B, the respective ROC curves in Figures 11A and 11B are not substantially (or significantly) different, indicating that CD103 is also highly specific for this subpopulation, and the slightly lower false positive rate for this three-gene panel indicates that further increasing the specificity of TRM identification will further improve its value as a biomarker. [Figure 11B] Same as above [Figure 12A] Figures 12A and 12B show the true-positive and false-positive prediction rates for Alzheimer's disease (AD) by CD103 alone from samples with the same exclusions as described for Figures 11D and 11E for the plots of the high T cell (Figure 12A) and low T cell (Figure 12B) populations. The accurate prediction of over 50% of AD patients, including less than 8% false-positive predictions in the high T cell samples, confirms that CD103 on T cells is relevant to AD and can serve as a single-gene blood biomarker for late-onset forms of the disease. [Figure 12B] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0020] All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology, 3rd Edition, Revised Edition, J. Wiley & Sons, Inc. (New York, NY, 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 7th Edition, J. Wiley & Sons, Inc. (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 those skilled in the art with a general guide to many of the terms used in this application. References for antibody preparation include D. Lane, Antibodies: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, NY, 2013); Kohler and Milstein (1976) Eur. J. Immunol. 6:511; Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988); U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Tomlinson I. and Holliger P. (2000) Methods Enzymol 326:461-479; Holliger P. (2005) Nat. Biotechnol. September 23(9):1126-36).

[0021] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For 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 a special receptor on the surface of the T cell, called the T cell receptor (TCR). Several different T cell subpopulations have been discovered, including T helper cells (T H cells), cytotoxic T cells (T C cells, or CTL), central memory T cells (T CM cells) and effector memory T cells (T EM memory T cells (T M cells), natural killer T cells (NKT cells), gamma delta T cells (γδT cells), and regulatory T cells (T reg Each of these has a different function, including the cells.

[0023] CD8+ T cells express the CD8 glycoprotein on their cell surface. C CD8+ T cells express TCRs that recognize specific antigens. Intracellular antigens (usually peptides generated by intracellular protein degradation) form complexes with MHC class I molecules and are then transported to the cell surface, where they are transported to the T cell surface. C It becomes recognizable by cells. C If the TCR of a cell is specific for that antigen, the T C The cell binds to the complex of the MHC molecule and the peptide, and the T C During this antigen-specific activation, T cells destroy the cells due to the affinity between CD8 and MHC molecules. C When CD8+ T cells are activated, their partner is a T CThey are recognized as cells and are generally classified as having a defined cytotoxic role 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 acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length). In other embodiments, APP peptides suitable for use with the aspects of the invention described herein may be derived from human APP having the full-length sequence set forth in SEQ ID NO: 1. In one embodiment, the APP peptide comprises the sequence 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). APP or APP peptides are further described in International Application Publication No. WO 2017 / 040594, the contents of which are incorporated herein by reference. In some embodiments, SEQ ID NOs: 2-6 represent readily manufacturable APP-derived peptides capable of stably binding to the most common HLA allele in the Western world (HLA-A2); however, other peptide-HLA combinations may be utilized depending on the anthropological characteristics of the patient cohort, as will be appreciated by those skilled in the art.

[0025] As used herein, "amino acid" is intended to include both naturally occurring and synthetic amino acids, and both D- and L-amino acids. A "standard amino acid" refers to any of the 20 L-amino acids commonly found in natural peptides. A "non-standard amino acid" refers to any amino acid other than a standard amino acid, whether synthetic or derived from natural sources. As used herein, "synthetic amino acid" also encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (e.g., amides), and substitutions. Amino acids contained within the peptides disclosed herein, particularly those at the carboxy or amino termini, can be modified by methylation, amidation, acetylation, or substitution with other chemical groups that can alter the circulating half-life of the peptides without adversely affecting their biological activity. Additionally, disulfide bonds may or may not be present in the peptides disclosed herein.

[0026] As used herein, the terms "peptide" and "protein" are used interchangeably to refer to a compound (e.g., a peptide isostere) composed of at least two amino acid residues covalently linked by a peptide bond or modified peptide bond. There is no limit to the maximum number of amino acids that may comprise a protein or peptide. It is understood that the amino acids comprising the peptides or proteins described herein and in the appended claims are either D-amino acids or L-amino acids, with L-amino acids being preferred. The amino acids comprising 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 anywhere in the peptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. It is understood that the same type of modification may be present to the same or varying degrees at several sites in a given peptide. A given peptide may also contain multiple types of modifications. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide derivative or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginine addition, and ubiquitination.See, for example, Proteins--Structure and Molecular Properties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; and Wold F., Posttranslational Protein Modifications: Perspectives and Prospects, pp. 1-12, in Posttranslational Covalent Modification of Proteins, BC Johnson (ed.), Academic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein cofactors," Meth. Enzymol. (1990) 182:626-646; and Rattan et al., (1992), "Protein Synthesis: Posttranslational Modifications and Aging," Ann NY Acad Sci. 663:48-62.

[0027] As used herein, a "sample" or "biological sample" refers to a tissue or body fluid removed from a mammal, preferably a human, that contains CD8 + Contains T cells or CD8 +The term "T cell" refers to a substance believed to contain T cells. The sample may be blood and / or a blood fraction, including a peripheral blood sample such as a peripheral blood mononuclear cell (PBMC) sample or blood (e.g., whole blood, plasma, serum), a bone marrow cell sample, or cerebrospinal fluid (CSF). The sample may be a brain tissue biopsy. The sample may include any specific tissue / organ of interest, including, but not limited to, lymphocytes, thymus, pancreas, eye, heart, liver, nerve, intestine, skin, muscle, cartilage, ligament, synovial fluid, and / or joint. These samples can be obtained from any individual, including healthy individuals or individuals with cells, tissues, and / or organs eliciting an unwanted immune response. Methods for obtaining such samples are well known to those skilled in the art of immunology and medicine. These methods include collecting and processing blood and blood components using routine procedures, or obtaining biopsies from bone marrow or other tissues or organs using standard medical techniques.

[0028] T cells are master regulators of inflammation throughout the body. Chronic inflammation is increasingly recognized as an important contributor to various human diseases, and misregulation of T cells promotes this chronic inflammation. Memory CD8 subsets undergo aberrant proliferation with age and increase in several tissues, including the brain. However, these proliferations occur only rarely in aging experimental animals, and their functional consequences are counteracted by persistent thymic activity.

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

[0030] Herein, we explore the age-related CD44 expression in rodent models to overcome the limitations of common experimental rodent models. hi CD123 + CD127 hi KLRG1 + CD103 +Homeostatic proliferation of CD8+ T cells is induced by infusion into athymic mice to obtain a resident memory phenotype. The resulting artificial homeostatically proliferating (hiT) cells not only exhibit signature age-related surface marker changes and TCR Vβ 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 As with cells, these artificial homeostatic proliferative resident memory ( hi T RM ) cells are abundant in the brain, where, surprisingly, they contribute to progressive neurological disease symptoms, including (i) increased APP cleavage products, (ii) diffuse beta-amyloid (Aβ) plaques in the brain, (iii) fibrillar inclusions in neurons, (iv) neuroinflammation, and (v) age-related cognitive impairment, while also contributing to the loss of neurons, synaptic markers, and brain mass. hi T cells exhibit pro-inflammatory functions that contribute to neurodegeneration and cognitive impairment in nude mice. Depletion of CD8+ T cells in the brain via CD103 deficiency suppresses age-related cognitive decline in immunocompetent mice. Furthermore, T cells are also expressed in both the brains of Alzheimer's disease and in the blood of patients with cognitive impairment. hi T RM The epitope specificity was altered, implicating this epitope in disease- 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, comprising a CD103 inhibitor, a CD8+ T RM and / or tolerogenic vaccines. As used herein, "prevention" includes, but is not limited to, reducing the likelihood of having the disease or condition or delaying its onset.

[0032] CD103 is also known as integrin αE, and in humans is encoded by the ITGAE gene, and is a member of the integrin α E The α subunit of β7 (also known as CD103) is an integrin protein. CD103 is a tissue-resident memory T (T) that is stably present in tissues such as the lung, gut, and peripheral non-lymphoid tissues, including the skin. RM ) cells, a subtype called memory CD8 + It limits T cells, where they orchestrate a local immune response that is highly protective against persistent viral infections.

[0033] In some embodiments, the CD103 inhibitor is an anti-CD103 antibody or an antigen-binding fragment thereof. Examples of anti-CD103 antibodies for the prevention or treatment of neurodegeneration include: (1) clone Ber-ACT 8 (2) a mouse anti-human CD103 monoclonal antibody (mAb) (BIORAD®) derived from clone 2G5.1 or a humanized version of 2G5.1; ​​(3) an anti-rat CD103 monoclonal antibody (mAb) OX-62 or a humanized version of OX-62; and (4) an anti-mouse CD103 monoclonal antibody (EBIOSCIENCE™) derived from clone 2E7 or a humanized version of 2E7.

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

[0035] In some embodiments, CD8+ T RMis administered to promote the treatment, suppression, reduction of the severity of, or prevention of age-related cognitive decline, pathological neurodegeneration, or both, and the effector molecule inhibitor is administered to promote the treatment, suppression, reduction of the severity of, or prevention of APP-specific CD8+ T cells. RM The therapeutic agent is a small molecule, antibody or antibody fragment, or nucleic acid that inhibits the activity or reduces the expression level of perforin 1, interferon gamma, or other inflammatory cytokines released by transformed effector T cells. According to one embodiment, a perforin 1 inhibitor is administered, including but not limited to diarylthiophenes and GSK2126458. According to another embodiment, an interferon gamma (IFNγ) inhibitor is administered, including but not limited to mesopram and rocaglamide.

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

[0037] Pharmaceutical Composition In various embodiments, the present invention provides a pharmaceutical composition for the prevention or treatment of age-related neurodegeneration, the pharmaceutical composition comprising a CD103 inhibitor, a CD8+ T RM and / or a tolerogenic 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 tolerogenic vaccine includes APP or an APP peptide, such as a peptide of SEQ ID NO:2-8.

[0038] The pharmaceutical compositions of the present invention can contain any pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" generally refers to an excipient that is safe, non-toxic, and useful in preparing the desired pharmaceutical composition, including excipients acceptable for use in human pharmaceuticals and veterinary medicine. Such excipients can be solid, liquid, semisolid, or, in the case of aerosol compositions, gaseous. Examples of excipients include, but are not limited to, starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, wetting agents, emulsifiers, colorants, release agents, coating materials, sweeteners, flavoring agents, fragrances, preservatives, antioxidants, plasticizers, gelling agents, thickeners, setting agents, setting and hardening agents, suspending agents, surfactants, humectants, 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 refer to any route of administration known in the art, including, but not limited to, aerosol, nasal, oral, transmucosal, transdermal, parenteral, or enteral. "Parenteral" refers to a route of administration generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, intrathecal, subcapsular, subcutaneous, transmucosal, or transtracheal. When administered via a parenteral route, the composition may be in the form of a solution or suspension for infusion or injection, or may be a lyophilized powder. When administered via a parenteral route, the composition may be in the form of a solution or suspension for infusion or injection. For enteral administration, the pharmaceutical composition may be in the form of controlled-release tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres, or lipid or polymeric vesicles. These compositions are typically administered by injection. These administration methods are known to those skilled in the art.

[0040] The pharmaceutical compositions of the present invention can include any pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle involved in the transport or delivery of a 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 with which it may come into contact, meaning that it must not pose a risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that significantly outweighs the therapeutic benefits of the carrier.

[0041] The pharmaceutical compositions of the present invention can be formulated into 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. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohol, and water. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia gum, agar, or gelatin. The carrier may also include a sustained-release material (e.g., glyceryl monostearate or glyceryl distearate), alone or with a wax.

[0042] Pharmaceutical preparations are made according to conventional pharmaceutical techniques, including grinding, mixing, granulating, and tableting as needed for tablet dosage forms, or grinding, mixing, and filling for hard gelatin capsule dosage forms. When a liquid carrier is used, the preparation is in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspension. Such liquid preparations can be administered orally or filled into soft gelatin capsules for administration.

[0043] The pharmaceutical compositions of the present invention may be delivered in a therapeutically effective amount. This precise therapeutically effective amount is the amount of the composition that produces the most effective results in terms of therapeutic efficacy in a given subject. This amount will vary depending on various factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, type and stage of disease, general health, response to a given dose, and type of drug), the nature of the pharmaceutically acceptable carrier or carrier in the formulation, and the route of administration. Those skilled in the medical and pharmacological fields can determine a therapeutically effective amount through routine experimentation, for example, by examining the subject's response to administration of the compound and adjusting the dosage accordingly. For additional guidance, see Remington, The Science and Practice of Pharmacy (Gennaro, ed., 20th ed., Williams & Wilkins, PA, USA) (2000).

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

[0045] After preparation of the liquid pharmaceutical composition, the liquid pharmaceutical composition may be lyophilized to prevent degradation and maintain sterility. Methods for lyophilizing liquid compositions are known to those skilled in the art. The composition may be reconstituted immediately prior to use with a sterile diluent (e.g., Ringer's solution, distilled water, or sterile saline), which may contain additional ingredients. Once reconstituted, the composition is administered to a subject using methods known to those skilled in the art.

[0046] kit In one embodiment, the kit comprises a step of isolating CD103-positive CD8+ T cells from a biological sample. RM The kit includes components necessary to identify, isolate, and / or enrich for a population of CD103+ CD8+ T cells. In another aspect of this embodiment, the kit may further include positive and / or negative controls and / or instructions for using the contents of the kit to identify, isolate, and / or enrich for CD103+ CD8+ T cells. In yet another aspect of this embodiment, the kit may further include culture vessels (e.g., dishes or flasks), media, or any necessary buffers or factors useful for promoting cell growth.

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

[0048] Instructions for use may be included in the kit. Typically, "instructions for use" include clear language describing techniques to be used in using the components of the kit to bring about a desired outcome, such as treating, reducing the severity of, inhibiting, or preventing age-related neurodegeneration in a subject. Optionally, the kit also includes other useful components, such as measuring devices, diluents, buffers, pharmaceutically acceptable carriers, syringes, or other useful tools as would be readily apparent to one of skill in the art.

[0049] According to various embodiments, CD8A-positive, CD44-positive, and CD103-positive CD8+ T cells RM Detection of CD8+ T cells RM The detection of the above other biomarkers is carried out using flow cytometry analysis based on fluorescence-activated cell sorting (FACS) technique. The detailed standard operating procedure for FACS flow cytometry analysis is provided in Example 2.

[0050] How to use A method for identifying a subject susceptible to or experiencing pathological neurodegeneration includes detecting CD103-positive resident memory CD8+ T cells (CD8+ T cells) in the peripheral blood of the subject. RM According to another embodiment, the subject is a human subject at least 65 years of age and is detecting an increased presence of CD8A-positive, CD44-positive, and CD103-positive CD8+ T cells in the blood. RM elevated levels of α-glucan are detected.

[0051] CD103-positive CD8+ T cells in subjects with memory impairment or age-related neurodegeneration RM a method for quantifying CD103-positive CD8+ T cells in a biological sample derived from the subject, RM Also provided are methods comprising detecting the amount of CD103-positive CD8+ T cells. In some embodiments, the methods include detecting the amount of CD103-positive CD8+ T cells. RM It further comprises comparing the amount of cells to a reference value.

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

[0053] The reference value for a quantitative assay or method can be a value obtained from a single control subject (e.g., a healthy subject without any symptoms of memory impairment or neurodegeneration) or a value obtained from a pool of multiple such control subjects. In other embodiments, the reference value is the subject's own value at a young age when they have no or few symptoms of memory impairment, and if a current value exceeds the reference value, it is used as a reference for determining high risk, the need for treatment for the condition, or suboptimal treatment outcome. 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 reference for determining the efficacy of the treatment.

[0054] Methods are provided for treating, inhibiting, reducing the severity of, or promoting the prevention of age-related cognitive decline, pathological neurodegeneration, or both in a subject in need thereof. The methods include administering a CD103 inhibitor, a resident memory CD8+ T cell (CD8+ T cell), or a combination thereof. RM ) and effector T cell inhibitors arising from CD8+ T RM and administering to the subject a therapeutically effective amount of one or more inhibitors of molecules released from the effector T cells arising from resident memory CD8+ T cells. In some embodiments, the CD103 inhibitor in this method is an anti-CD103 antibody, and the inhibitor of effector T cells arising from resident memory CD8+ T cells includes an inhibitor of perforin 1 or an inhibitor of IFNγ. According to one aspect, this administration results in CD8+ T cell activation to an APP peptide (e.g., the peptide of SEQ ID NO: 8). RMor effector T cell responses or binding derived therefrom are reduced compared to those obtained in a single control subject (e.g., a single healthy subject without any symptoms of memory impairment or neurodegeneration) or compared to those obtained from a pool of multiple such control subjects. According to another embodiment, the administration results in a reduction in CD8+ T cell responses to the APP peptide (e.g., the peptide of SEQ ID NO: 8). RM Or the response or binding of effector T cells derived therefrom is reduced compared to the values ​​in the subject when he / she is young and shows no or almost no symptoms of memory impairment.

[0055] Also provided is a method for treating, inhibiting, reducing the severity of, or promoting the prevention of age-related cognitive decline, pathological neurodegeneration, or both in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a tolerogenic vaccine that delivers an amyloid precursor protein or a peptide fragment thereof (e.g., any of the peptides set forth in SEQ ID NOS: 2-8).

[0056] A method for identifying a human subject who is susceptible to or experiencing age-related cognitive decline or pathological neurodegeneration, comprising measuring the presence or absence of CD103+ resident memory CD8+ T cells (CD8+ T cells) in a blood sample obtained from the human subject who is experiencing one or more of the following symptoms: loss of short-term or long-term memory, impaired ability to maintain concentration, and impaired problem-solving ability. RM According to one embodiment of the method, the method comprises detecting an increased presence of CD103+CD8+ T RM The increased presence of is compared to a value obtained from a healthy human subject or a pool of healthy human subjects who do not have any of the above symptoms. According to another embodiment, the 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 methods is a human. In some embodiments, the human subject is middle-aged or older, e.g., after 30 years of age, after 35 years of age, after 40 years of age, after 45 years of age, after 50 years of age, after 55 years of age, after 60 years of age, after 65 years of age, after 70 years of age, after 75 years of age, after 80 years of age, after 85 years of age, after 90 years of age, or after 95 years of age. In other embodiments, the human subject has previously been treated with CD103-positive CD8+ T cells. RM I have shown cell recordings.

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

[0059] Animal models, etc. A system for identifying and / or screening candidate therapeutic, preventative, and / or diagnostic agents for cognitive decline in humans, comprising: a CD44 antibody derived from a rodent (e.g., a mouse); hi CD123 + CD127 hi KLRG1 + CD103 + The above system is provided comprising a resident memory CD8+ T cell phenotype. In some embodiments, this CD44 hi CD123 + CD127 hi KLRG1 + CD103 + The resident memory CD8+ T cell phenotype is achieved by administering resident memory CD8+ T cells to athymic mice.

[0060] A method for identifying and / or screening candidate agents for the treatment or prevention of age-related neurodegeneration in humans includes screening candidate agents in vitro for CD44 hi CD123 + CD127 hi KLRG1 + CD103 +contact with resident memory CD8+ T cells or CD44 hi CD123 + CD127 hi KLRG1 + CD103 + The method includes administering a candidate agent to a model animal containing resident memory CD8+ T cells, and identifying a reduced level of CD103-positive resident memory CD8+ T cells, a reduced level of effector molecules on these cells, or a reduced amount of CD8+ T cells migrating from the periphery to the brain of the animal. [Example]

[0061] The following examples are presented to better illustrate the invention claimed in this application and should not be construed as limiting the scope of the invention. Reference to specific materials is for illustrative purposes only and is not intended to limit the invention. Those skilled in the art may develop equivalent methods or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0062] Example 1. Aberrant resident 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, occurring gradually with aging but also rapidly upon infusion into young, T cell-deficient hosts (20, 21). Given that this phenomenon has been demonstrated to be associated with age-related CD8+ T cell dysfunction, this inducible phenomenon allows for unambiguous validation of the role of abnormal CD8+ T cells in diseases such as Alzheimer's disease. Spontaneous induction of homeostasis by infusion into nude mice consistently induced CD8+ T (hiT) cells that displayed molecular, phenotypic, and functional abnormalities indistinguishable from those observed in diseased aging mice. These hiT cells localized to the brain, where they ultimately promoted Alzheimer's disease-like neurodegenerative symptoms, including prominent disease features that were lost in FAD mutant transgenic animals. hiT cell-related endpoints were also elevated in human Alzheimer's disease brains. Our studies identify age-related immune cell processes that overcome resistance in mice to age- and risk factor-induced Alzheimer's disease-like symptoms. These findings have important implications for modeling age-related diseases in mice, as well as for investigating the pathogenesis and treatment of sporadic Alzheimer's disease.

[0063] Materials and Methods Animal subjects Female C57BL / 6 and B6.Foxn1 mice, as well as congenic and / or isogenic knockout strains (Jackson Laboratories), were housed in a pathogen-free animal facility under standard conditions with a 12-hour light / 12-hour dark cycle, with food and drink ad libitum. Recipient animals were 8-10 week-old female B6.Foxn1 mice (n > 5), B6.Foxn1-AppKO mice (n > 4), or B6.CD45.1 congenic mice (n > 5). Donors were 5-8 week-old females of the same strain. Cell sources were randomized by pooling more than five donors per experiment. Young (8-10 weeks) and aged (15 months) male and female C57BL / 6 and B6.CD103-knockout mice (young mice, n = 12; aged mice, n = 7-8) were used to study age-related cognitive decline. Donor, recipient, and naive animals were housed in a pathogen-free facility in the Department of Comparative Medicine at Cedars-Sinai Medical Center, and all breeding and genetic screening was performed at Jackson Laboratories (Bar Harbor, ME).

[0064] Adoptive transfer of CD8+ T cells Splenic CD8 T cells from female C57BL / 6J mice (5–7 weeks old) were purified using anti-CD8 immunobeads (Miltenyi Biotec, Sunnyvale, CA). 3 × 10 cells in 50 μl of PBS were cultured. 6 CD8+ T cells were intravenously injected into female C57BL / 6J or B6.Foxn1 nude mouse hosts. Efficiency of transfer into B6.Foxn1 hosts was verified by the retention of CD8+ T cells in splenic lymphocytes at greater than 5% 3 weeks after injection. The order of treatments was randomized by alternating cell and control injections among individual recipients. For all subsequent analyses, both the identity of the groups and the expected results were concealed from the investigators performing the analyses.

[0065] Tissue (brain, spleen) processing Brains and spleens were harvested from PBS-perfused mice. 1 mm sections were prepared from the brain to the right of the intertemporal fissure (midline). For protein studies, the right hemisphere was snap-frozen at -80°C and subsequently homogenized in cell lysis buffer (Cell Signaling Technologies, MA), followed by centrifugation of cell nuclei. Cell lysates were separated into Triton-soluble, sarkosyl-soluble, and sarkosyl-insoluble fractions using sequential incubations with 10% (wt / vol) salt-sucrose solution and 1% (wt / vol) sarkosyl 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 weighed on a Mettler balance, removing the cerebellum, brainstem, and olfactory bulbs.

[0066] Western blot Triton-soluble cell lysates were separated by electrophoresis on 12% Tris-HCl precast gels (Bio-Rad) and blotted onto 0.2 μm nitrocellulose. Membranes were blocked with BSA, incubated in sequential dilutions of primary and secondary antibodies for 1 h at room temperature, washed three times, developed using enhanced chemiluminescence substrate (GE Healthcare Biosciences, Pittsburgh, PA), and exposed to Amersham Hyperfilm (GE Healthcare Biosciences, Pittsburgh, PA).

[0067] ELISA The supernatant of homogenized brain tissue was used for the Triton-soluble fraction of Aβ. The insoluble pellet from Triton-homogenized brain was resuspended in 10x the volume of 5 M guanidine hydrochloride for 4 hours to generate guanidine-soluble Aβ. Triton-soluble and guanidine-soluble samples were analyzed by soluble and insoluble Aβ ELISA (Invitrogen, Life Technologies; Grand Island, NY). Absorbance was read on a SPECTRAmax Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA), and delta was analyzed using Graphpad PRISM (GraphPad Software, San Diego, CA).

[0068] Flow cytometry Purified T cells stained with each antibody were analyzed by three-color flow cytometry (FACScan II; BD Hi-O Sciences, San Jose, CA) to assess purity. Whole spleen single-cell suspensions in PBS containing 5% FBS were incubated with the antibody for 30 minutes on ice, followed by a wash with PBS containing 5% FBS. 100,000–300,000 flow events were acquired.

[0069] Antibodies for tissue staining and Western staining Free-floating brain sections (8–14 μm thick) were mounted on slides and blocked for 1 hour at room temperature. Sections were incubated with primary antibodies in blocking solution (Dako, CA) overnight at 4°C. Sections were washed four times in PBS and incubated for 90 minutes with fluorochrome- or biotin-conjugated secondary antibodies with or without curcumin (0.01% in PBS), or with thioflavin S alone (1% in PBS). Sections were washed, coverslipped, and mounted with ProLongGold antifade media containing DAPI (Invitrogen). Brightfield and fluorescent images were acquired using a Zeiss AxioImagerZ1 (Carl Zeiss Microimaging) equipped with a CCD camera. Image analysis of the microscopic images was performed using ImageJ (NIH). Anti-Aβ / APP antibody (Abcam ab14220 for the 3-week time point; Chemicon clone 4G8 for all others) was used at 1:500 for immunohistochemistry (IHC) and 1:1000 for Western blot (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 PHFs. For marker size, p-tau WB signals were normalized to the signal of β-actin (clone AC-74, Sigma), and GAPDH was used for normalization of 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 at 1:200 for IHC. Anti-CD8 (clone 53-6.72, BD Pharmingen) was used at 1:100 for IHC and 1:1000 for WB. All secondary antibodies (HRP, AlexaFlour-488, -594, -647; Invitrogen) were used at 1:200 for IHC and 1:2000 for WB.Multimer generation and use: Dextramers of epitopes engineered for autoantigens / brain antigens (Trp-2-DCT(180-188) / H-2Kb) and / or custom APP epitopes with predicted affinities less than 100 nM (NetMHC version 3.4) were generated by Immudex.

[0070] Gallias silver staining Gallias silver staining was used to visualize fibrillar aggregates. Free-floating brain sections were placed in 5% periodic acid for 3 minutes, washed twice, placed in silver iodide solution for 1 minute, then 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 a light brown / gray color. The color development was stopped by placing the sections in 0.5% acetic acid for 5 minutes, washed with distilled water, and mounted on slides. Stained sections were examined microscopically. Stained neurons were counted in the CA2 region of the hippocampus, and the number of stained neurons among the total neurons in the entorhinal and cingulate cortices was visually quantified in triplicate experiments.

[0071] Neuron counting Total neuronal cell counts were estimated using an optical sectioning method with stereological analysis software (Stereo Investigator; MBF Biosciences). Paramedian sagittal serial sections spaced 50 μm apart were stained for NeuN. CA1, CA2, CA3, and other regions of interest were defined based on the Paxinos and Watson mouse brain atlas. A grid was randomly placed over the ROI, and the number of cells within a three-dimensional optical dissector (50 μm × 50 μm × 10 μm) was counted using a 100x objective. Within each dissector, 1 μm guard zones were excluded on the top and bottom of the section. Using the Stereo Investigator software, an estimated total count was obtained, weighted by section thickness, resulting in an error coefficient of 0.10.

[0072] Behavioral testing At 3, 6, and 13 months after cell or control injection, open field testing was performed before all other behavioral testing. At 6 and 11 months after cell or control injection, the number of flinch jumps / conditioning freezing behaviors was determined. At 12 months after cell or control injection, mice were tested for SA only once. At 14 months after cell or control injection, a single Barnes maze test was performed. The order of behavioral testing was randomized by alternating experiments with control and treatment animals. For tests conducted on more than one day, testing began simultaneously (±1.5 h), and early and late testing times were alternated for randomization between groups. Additional randomization was employed in the Barnes maze by alternating the location of the escape compartment between animals per group and between each of three daily training trials per animal.

[0073] Barnes maze (BM) test The Barnes maze is a spatial learning task that allows subjects to use spatial cues to locate a means of escape from a mildly aversive environment (i.e., mice are required to use spatial cues to find a refuge). Mice were assessed for their ability to learn the location of the escape box over a 9-day period in the Barnes maze 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 (three trials), followed by two days without testing and a retest on the 7th day. Each trial was separated by a 35-60 min intertrial interval. Each trial began with placing a mouse into the start box, a bottomless cube located in the center of the maze. After 30 s, the start box was lifted, and the mouse was released from the start box to find the escape hole. Two fluorescent lights located on the ceiling or high in the room illuminated the testing chamber. Each trial lasted a maximum of 4 min, or until the mouse entered the escape box. After each training trial, the experimenter guided mice that failed to find the escape hole within 4 minutes to the correct hole. Once the mouse entered the escape box, it was allowed to remain in the box for 1 minute. After the 7th day of testing, but on different days, mice were tested for two more days, in which the escape box was reversed on the 8th day and returned to its original position on the 9th day. The exact same testing procedure was applied to all mice in all groups. After each test and before each day's testing, the maze and all compartments were thoroughly cleaned with isopropyl alcohol to remove any olfactory cues.

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

[0075] Flinch jump / fear conditioning test First, a flinch jump test was used to determine whether there were significant differences in nociceptive thresholds (pain sensitivity) between treatment groups. Subsequently, Pavlovian fear conditioning was used to assess learning and memory for aversive events. The apparatus (Freeze Monitor™, San Diego Instruments, San Diego, CA) consisted of a Plexiglas box (25.4 × 25.4 × 31.75 cm high) with a stainless steel grid floor. An audio stimulus unit was placed on top of the box, and light beams and optical sensors were positioned around the box. These optical sensors were connected to a computer via an input matrix, which automatically recorded interruptions of the light beam. For testing, on day 1, individual mice were placed in the test box and allowed to habituate for 3 minutes. At the third minute, a tone was presented for 30 seconds. Thirty seconds after the tone ceased, a 0.5-second foot stimulation (intensity = mean jump threshold for that treatment group as determined by the flinch jump test) was administered. The mice were then removed from the box and returned to their home cages for 2 minutes. The chamber was cleaned, the animal returned to the chamber, and the procedure was repeated. The number of freezing events (5 seconds without abnormal movement and no interruption of the light beam) during this procedure was recorded using a freezing monitor. On the second day, mice were placed in the same test box where they had previously received the sound and foot stimuli to determine whether they recalled the situation, but this time without the sound and foot stimuli. The number of freezing events was measured over a 10-minute period. On the third day, cue conditioning was measured after placing a triangular Plexiglas box inside the test box. The mouse was placed in the triangular chamber, where it had not previously received the sound or foot stimuli. One minute later, a 30-second sound was presented, and the number of freezing events was measured over a 10-minute period. All data from the flinch-jump and fear conditioning tests were first normalized to the mean of the first two training trials on day 1 within each group, and then normalized to the contextually learned freezing value of the PBS control or the cue-learned freezing value within all experimental groups, expressed as percent of control, and analyzed by ANOVA followed by the Newman-Keuls test, where appropriate, to detect differences between treatment groups.

[0076] Open field test The test was conducted in an open-field apparatus consisting of an open-topped, clear Plexiglas box measuring 16" x 16" x 15" high. Two ring-shaped light beams and optical sensors were placed around the periphery of the box. The optical sensors were connected to a computer by an input matrix. Each mouse was placed in the box, and light beam interruptions were automatically recorded and used as a measure of locomotor activity. Each mouse was tested in the box for a 30-minute period.

[0077] statistical analysis The number or area (μm ) of β-amyloid plaques, GFAP+, Iba1+, or Perforin1+ cells was measured in 6–8 coronal sections from each individual at 150 μm intervals (unless otherwise indicated) across a 900–1200 μm area encompassing the hippocampus and cortex. 2 Quantitative and stereological counting of the chromatin was analyzed. Specific fluorescent signals were captured with the same exposure time for each image, and optical sections of each field of the specimen were entered into NIH ImageJ and analyzed as described above. GraphPad Prism (version 5.0b; San Diego, CA, USA) was used for data analysis using ANOVA and Welch's corrected t-test (equal variances not assumed). All histograms show the mean + SEM.

[0078] The sample sizes for the PrfKO-CD8 and IfnγKO-CD8 groups were calculated a priori for each evaluation index using the mean and standard deviation of the PBS and wild-type CD8 groups for the expected effect size, with an alpha of 0.05 and a confidence level of greater than 95. The calculated n plus any number greater than 1 was then used for the PrfKO-CD8 and IfnγKO-CD8 groups.

[0079] Sections or samples without discernible background signal and values ​​within each group that were more than two standard deviations above or below the median were excluded. The number of subjects and reagent validation methods are listed in Table S1.

[0080] Study Approval All animal experimental procedures were approved by the Cedars-Sinai Medical Center Institutional Review Board prior to their implementation. The Cedars-Sinai Medical Center Institutional Review Board designated the analysis of de-identified human brain specimens from the University of California, Davis, as exempt from committee review. Brain specimens were collected, stored, and distributed with prior approval by the University of California, Davis Medical Center Institutional Review Board.

[0081] result Generation of "hiT" cells in nude mice CD8+ T cells from young (<9 weeks) C57BL / B6 (B6) donors were infused into B6.Foxn1 recipients and subjected to phenotypic analysis (Figures 1A, 1H, and 1I). Donor CD8+ T cells rapidly expanded in the blood of young B6.Foxn1 recipients within 3 days, and these cells persisted in the blood for an extended period (Figures 1J and 1K). Nude mouse hosts were transfected with wild-type B6 or B6.CD45.2 congenic [B6 (Cg) ] CD8+ T cells serially transferred into the host did not further expand (Figure 1I and 1K). Analysis of artificially homeostatically expanded donor CD8+ T cells ("hiT" cells) in B6.Foxn1 hosts showed a surface marker profile identical to that of CD8+ T cells undergoing clonal expansion in aged mice (CD122 hi , CD127 hi , CD44 hi , KLRG1 hi , PNA hi , CD8 lo , CD103 +(Figure 1A-1D). A similar phenotype is observed in clonal expansion of CD8+ T cells in aging humans. Accordingly, CFSE-labeled CD8+ T cells exhibited a ladder-like dye 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), indicating that rapid homeostatic proliferation may depend on responsiveness to APP.

[0082] To examine the clonality of hiT cells, we analyzed variable region D→J rearrangements 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 skewing of TCR Vβ chains, whereas peripheral T cells infused into nude mouse recipients showed D1→J1 and D2→J2 clonal skewing after just 10 weeks (Figures 2F and 2G). Importantly, in contrast to the diverse D→J usage seen in young wild-type mice, D1→J1 and D2→J2 clonal skewing was also evident in the brains of young nude mice infused with CD8+ T cells (Figures 1E and 1F). This pattern most closely resembled D→J usage in the brains of aged mice.

[0083] CFSE-labeled donor CD8+ T cells were enriched in the brain parenchyma 3 days after intravenous infusion in B6.Foxn1 mice, directly demonstrating rapid homing of hiT cells to the brain (Figures 2A and 2B). Flow cytometry revealed only a modest increase in total CD8+ T cells 10 weeks after infusion in B6.Foxn1 mice compared with wild-type B6 mice (Figure 2C). Western blot analysis revealed a clear increase in CD8 protein at this time point, suggesting increased cell influx without an increase in viable cells (Figure 2H). Indeed, flow cytometry revealed that IFNγ-treated mice retained CD103 expression. + CD8+ T cells and KLRG1 +Both CD8+ T cells were significantly increased in the brains of nude mouse recipients at this time point, indicating a qualitative, rather than quantitative, change in CD8+ T cells in the brain (Fig. 2C). + CD8+ T cells reacted to MHC class I-restricted 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 reactive to APP epitopes selectively accumulated in the brains of nude mice, we decided to analyze APP-related symptoms (Figure 3K).

[0084] Aβ and neurofibrillary deposition Western blot analysis revealed that detergent-soluble APP and derived cleavage products (APPs) were expressed in the excised cortex and hippocampus of B6.Foxn1 hosts 3 and 10 weeks after intravenous CD8+ T cell infusion. Cl ) was increased (Figures 3A and 3I). Aβ1-40 was elevated by ELISA at 2.5 months and remained elevated at 15 months (Figure 3B), and increased Aβ was observed in the vasculature 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 (mice in the wild-type CD8 group) injected with wild-type CD8 T cells at 15 months (Figures 3C and 3N). However, unlike mice expressing familial genetic mutations seen in human Alzheimer's disease, in hiT-bearing nude mice, Aβ1-42 was not significantly altered, and amyloid plaques were primarily diffuse and showed little co-staining with curcumin or thioflavin S (Figures 3C and 3O). Thus, the amyloidopathy in hiT-bearing nude mice differed from that seen in the ADtg mouse model.

[0085] Curcumin and thioflavin S stained cells within the dentate gyrus of wild-type CD8 mice 6 months after T cell infusion (Figure 3K). Similar structures were not observed in aged ADtg mice (Figure 3K) or ADtg rats, which exhibited tau paired helical filaments (Figure 3L). This indicated that hiT-bearing nude mice may have fibrillar inclusions composed of hyperphosphorylated tau protein in neurons. Thus, 10 weeks after infusion, the brains of wild-type CD8-bearing mice showed a ∼30% increase in p-tau in the Triton-soluble fraction, and a nearly 5-fold increase in larger tau PHFs (Figures 3E and 3F). The increase in p-tau was not sustained, but tau PHFs remained 2.5-fold higher than controls at 15 months after infusion (Figure 3F). Most interestingly, silver-stained cells also increased in the hippocampus, entorhinal cortex, and cingulate cortex of wild-type CD8 mice at this time point (Figure 3G, H). By sequential silver / immunofluorescence staining, these cells were identified as nuclear p-tau cells. + Although these lesions were indicative of neuronal origin, no anuclear "ghost tangles" were observed (Figures 3G, 3M, and 3N). Concomitantly stained ADtg mouse brains showed only silver-stained plaques (Tg2576 mice; Figure 3G), confirming silver-stained cells exclusively in hiT-bearing mice. These data indicate that hiT cells promote the organized deposition of Aβ40 in the parenchyma, diffuse plaques, and fibrillar inclusions in live neurons.

[0086] Immune and neuroinflammatory infiltrates Although not evident from forebrain flow cytometry, the number of CD8+ T cells was significantly increased in hippocampal slices from mice in the wild-type CD8 group 15 months after injection, and they happened to express p-tau. + The number of CD8+ T cells did not increase outside the hippocampus. Iba1 in the cortex and hippocampus + Microglia and activated GFAP +Astrocytes were also significantly increased in wild-type CD8+ mice compared with controls (Figures 3I and 3J). Aβ plaque area was more strongly correlated with hippocampal CD8+ T cell numbers than with cortical or hippocampal astroglial proliferation, consistent with a strong influence of T cells on amyloidopathy (Figures 3Q, 3R, and 3S).

[0087] Neuronal loss and brain atrophy Fifteen months after T cell infusion, NeuN in the CA2 of wild-type CD8 mice compared with controls + The number of T cells decreased (Figures 4A-C). Furthermore, 6 months after T cell infusion, the wild-type CD8 group experienced a 5% decrease in brain volume, which progressed to a 10% decrease at 15 months (Figure 4D). Significant neuronal and synaptic loss in the wild-type CD8 group was confirmed by Western blot reductions of NeuN, drebrin, and synaptophysin signals, each of which showed approximately a 10% signal decrease at 15 months after infusion (Figures 4E and 4F). The NeuN Western blot signal significantly correlated with brain volume across 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 treated and control nude mouse recipients at 3, 6, or 13 months after T cell infusion (Figure 4H). In contrast, wild-type CD8 mice showed a specific reduction in fear-conditioned responses to contextual learning 6 months after T cell infusion, and at 11 months, responses to both contextual and cued learning were reduced (Figure 4I). These results suggest that cognitive impairment in the wild-type CD8 group was limited to hippocampal function (required for contextual FC) in the early stages, but impairs both hippocampal and amygdala function (required for cued FC) in the advanced, late stages. A similar pattern of progressive cognitive deficits also occurs in human Alzheimer's disease. Contextual learning performance at 6 months also correlated with brain volume, indicating its relevance to neurodegeneration.

[0089] To independently confirm cognitive deficits, we measured spontaneous alternation behavior 12 months after injection. This test is based on the mice's preference to alternate between exploring two paths and requires memory for the previously visited path. A minimum possible score of 50% indicates random path selection 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 published wild-type values ​​(27), whereas the SA of the wild-type CD8 group was 50% (Figure 4J). To test whether this reflected a memory or preference deficit, we performed the Barnes maze test, a definitive assessment of hippocampal-dependent memory and learning, at 14 months. Wild-type CD8 nude mice showed no improvement in learning this maze over the first 4-day training period, while all other groups showed significant improvement (Figure 4K). Given this early deficit, wild-type CD8 mice were expected to also have deficits in the retention and reversal learning phases of this maze (Fig. 4L-N). As with fear conditioning, there was a significant correlation between Barnes maze performance and brain volume. Thus, wild-type CD8 nude mice showed progressive, severe, and persistent learning and memory impairments without obvious motor deficits.

[0090] We further investigated whether Barnes maze performance was associated with elevated tau and / or Aβ outcome indices. Poor Barnes maze performance (total latency below median = BM) was associated with elevated tau and / or Aβ outcome indices. lo ) was not significantly associated with increased soluble p-tau, but was significantly associated with increased tau PHFs by Western blotting. In contrast, poor maze performance was not significantly associated with either Triton-soluble Aβ40 / Aβ42 or guanidine hydrochloride-soluble Aβ40 / Aβ42 by ELISA. Thus, as reported in human Alzheimer's disease, tauopathy rather than amyloidopathy reflected cognitive impairment in wild-type CD8 nude mice.

[0091] Cellular mechanisms of hiT cell-mediated neurodegenerative conditions To determine the mechanisms involved in hiT cell-mediated neurodegenerative symptoms, we infused CD8+ T cells from knockout donors deficient in perforin 1 or IFNγ, key effectors of T cell lytic and proinflammatory activity, respectively, into B6.Foxn1 mice. CD8+ T cells deficient in either gene (Prf1 or Ifnγ, respectively) proliferated comparable 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 and Ifnγ-KO-CD8, respectively) showed increases in soluble Aβ or p-tau / PHF at any time point (Figure 3B, Figure 3F). However, the IfnγKO-CD8 mice showed only slightly reduced accumulation of amyloid plaques and silver-stained cells in the hippocampus and entorhinal cortex compared with the wild-type CD8 mice, but these did not extend to the cingulate cortex (Figures 3D and 3H). The IfnγKO-CD8 mice also showed reduced astroglial and microglial proliferation (Figures 3I and 3J). However, unlike the PrfKO-CD8 mice, CD8+ T cells were present in significant numbers in the brain 15 months after injection (Figures 3O and 3P). The IfnγKO-CD8 mice also showed significantly reduced brain mass and NeuN levels at 15 months after injection. + Unexpectedly, both PrfKO-CD8 and IfnγKO-CD8 mice showed significant increases in both CD8 T cells and cerebral cortex. Finally, both PrfKO-CD8 and IfnγKO-CD8 mice showed significantly reduced cognitive function at 11–15 months. Thus, PrfKO-CD8 mice showed no evidence of pathophysiology by any criterion, including an increase in CD8 T cells in the brain, whereas IfnγKO-CD8 mice maintained some molecular pathophysiology, although they showed no evidence of neurodegeneration or cognitive decline.

[0092] CD8+T in age-related cognitive decline RM CD8+T RM Although CD103 induces neurocognitive symptoms in nude mice, it was unclear whether these cells also mediate age-related neuronal defects in immunocompetent mice.RM Its expression is characteristic of T RM We demonstrate that genetic deficiency of CD103 primarily affected CD8+ T cells (Fig. 5A) and reduced brain CD8 content (Fig. 5B, 5C). Young and aged CD103-deficient mice performed similarly to their wild-type counterparts during the Barnes maze training phase, despite a decline in locomotor activity with age (Fig. 5D, 5E). In contrast, aged CD103-deficient mice performed slightly better during the memory retention and reversal learning phases (Fig. 5F, 5G) and made significantly fewer errors in the Barnes maze, reversing the age-related differences documented in this strain of mice (Fig. 5H). Thus, CD103 deficiency protected aged mice from age-related cognitive decline. CD103 deficiency primarily affected CD8+ T cells and reduced CD103 + Only CD103 cells increase with age, either inside or outside the brain, suggesting that this may contribute to cognitive decline during aging. + CD8+T RM This confirms the involvement of CD8+ T cells, as age-related cognitive decline is a strong predictor of future neurodegenerative conditions. RM is further associated with disease dementia (e.g., AD).

[0093] hiT cell phenotype, effector proteins, and specificity in the human Alzheimer's disease brain To explore the possible relevance of hiT cells to human Alzheimer's disease, we focused on perforin 1 and CD8 in the brains of these patients, since IFNγ is already known to be associated with disease risk. Western analysis demonstrated the expected antibody specificity (68-75 kDa Prf1, 33-35 kDa CD8α) and the expected punctate pattern of anti-Prf1-stained lymphocyte nuclei (Figure 6C). Western signals for perforin 1 and CD8 correlated (n = 6; r = 0.8155, P = 0.048), and both were increased in the cortex of patients with mild, but not severe, Alzheimer's disease, with perforin 1 reaching statistical significance (Figure 6C). The perforin 1:CD8 signal ratio was also significantly increased in the brains of patients with severe Alzheimer's disease, consistent with the long-term qualitative changes in lymphocyte lysate composition observed in hiT cell-bearing mice (Figure 6B). To further investigate this, we investigated the T cell epitope [APP (471-479) We generated pHLA-A2 multimers against a human T cell epitope similar to APP. Hippocampal sections from patients with severe Alzheimer's disease and normal aging were stained with anti-CD8 and this or a control multimer to quantify the percentage of epitope-reactive CD8+ T cells. Subtracting staining from the negative control, we found that APP (471-479) CD8+ T cell reactivity to APP was significantly elevated in the diseased brains (Figures 6E and 6F; P = 0.002). While total CD8+ T cell levels were not significantly elevated in the diseased brains as in hiT cell-bearing mice, they were slightly elevated (n = 10; 1.6 ± 0.29 vs. 2.3 ± 0.55, P = 0.31). Thus, APP-reactive CD8+ T cells were elevated in the brains of severe Alzheimer's disease, similar to those in the brains of hiT cell-bearing nude mice.

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

[0095] Nude mice bearing hiT cells showed several similarities to human neurodegenerative disorders, particularly Alzheimer's disease. Specifically, early Aβ accumulation and late amyloid plaque accumulation, as well as neuroinflammation, silver-staining (fibrillar) neuronal inclusions, synaptic and neuronal loss with brain atrophy, and progressive cognitive impairment, were evident in these mice. Some of these features were not observed in mice expressing only the familial Alzheimer's disease gene mutation. Most notably, these included neuronal loss with brain atrophy and neurofibrillary inclusions. However, there were differences between the neurological symptoms of mouse models of Alzheimer's disease or FAD and those of nude mice bearing hiT cells. Aβ40 was clearly increased without Aβ42, and the amyloid plaques were predominantly 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 APP mutation exhibited a pattern of predominantly elevated Aβ40, a feature that distinguished hiT cell-bearing nude mice from most transgenic models as well as most human Alzheimer's disease models. This may be due in part to the prominent vascular amyloid in hiT cell-bearing nude mice, whose predominant Aβ40 composition may mask other Aβ species. Mouse Aβ40, while showing the opposite pattern to human Aβ peptides, also possesses a reduced efflux rate relative to Aβ42, which is expected to promote greater retention of Aβ40. Factors that specifically inhibit Aβ42 fibril assembly in rodent brains may further promote the dominance of Aβ40. Ghost tangles, on the other hand, differ from other neurofibrillary structures in that they are primarily composed of 3R tau, which is virtually absent in adult mice. Therefore, the discrepancy between hiT-bearing 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-cell-bearing nude mice progressing from hippocampus-dependent 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 is not surprising that increases in both CD8 and perforin-1 were observed in the brains of early Alzheimer's disease, similar to the increase in effector CD8+ T cells in hiT-bearing nude mice. Furthermore, Appl Pharmacology (2019) 24:119–120 1 3 119 119 129 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 610 620 630 640 650 660 670 680 690 700 710 720 730 740 750 760 770 780 790 800 810 820 840 850 860 87 [471-479] Increased CD8+ T cell reactivity to a nearly identical epitope (App [470-478] ) was directly proportional to the proliferation of CD8+ T cells responding to the antigen.

[0097] Lytic and proinflammatory T cell effector functions differentially affected the neuropathological features of hiT cell-bearing mice. Perforin-1 deficiency suppressed all neuropathological and symptomatic features, as well as CD8+ T cell retention in the brain. In contrast, Ifnγ deficiency permitted the accumulation of amyloid and neurofibrillary structures, albeit in a limited distribution reminiscent of early preclinical stages of Alzheimer's disease. This indicates that IFNγ promotes disease pathology, as previously reported. The defects in astroglial and microglial proliferation in IfnγKO-CD8 mice are also consistent with earlier studies showing that IFNγ promotes neuroinflammation in distinct Alzheimer's disease models. However, the unexpected increases in NeuN, drebrin, and brain mass in IfnγKO-CD8 mice suggest that IFNγ may also regulate neurodegeneration independently of neuroinflammation. Considering these differential effects, perforin-1 + or IFNγ + It will be interesting to determine whether these CD8+ T cells, respectively, are biomarkers 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 key role in the pathogenesis of neurodegeneration. Furthermore, the recent discovery of lymphatic vasculature in the brain suggests a possible structural basis for the general involvement of T cells in brain pathophysiology. While some, but not all, previous studies have reported an increase in CD8+ T cells or common autoimmune disease features in Alzheimer's disease, the nature of T cell involvement remains unclear. While it is reasonable to speculate that lytic autoreactivity may contribute to hiT cell-induced neurological symptoms, these symptoms differ in several ways from the classic autoimmune neurodegeneration characteristic of multiple sclerosis or experimental autoimmune encephalomyelitis. For example, these neurological symptoms were dependent on CD8+ T cells rather than CD4+ T cells, were ameliorated rather than exacerbated by IFNγ deficiency, and were not accompanied by dense immune infiltration. Furthermore, hiT-carrying mice lacked the distinct motor deficits of MS and EAE. This, together with the Alzheimer's disease-like neurological symptoms, strongly indicates that hiT cell-bearing nude mice do not exhibit MS-like autoimmune neurodegeneration.

[0099] It is possible that hiT cell-mediated neurological symptoms may cooperate with FAD mutations to produce a truly complete Alzheimer's disease-like pathological and symptomatic profile. It will also be important to question whether this complete disease-like feature in hiT cell-bearing mice can be obtained in human Aβ (App) and / or tau (Mapt) knock-in lines, and the overall role of strain background in modulating neurological symptoms. Most importantly, confirming the association of hiT cells with sporadic Alzheimer's disease, its prodromal states, and risk factors is crucial given their potential as biomarkers and etiological implications. Our findings tentatively demonstrate that hiT cells overcome the normal resistance of mice to the onset of age-related Alzheimer's disease-like neurodegeneration and the development of neurofibrillary inclusions. This is the first isolated physiological factor directly promoting age-related Alzheimer's disease-like neurodegeneration and the first immune cell hallmark of aging. The findings herein are the first evidence that abnormal CD8+ T cells promote tissue degeneration in an age-related disease state. It is conceivable that hiT cells reactive to different tissue antigens may cause damage to the brain or other regions of the body. Therefore, this hiT model and age-related CD8+ T cell dysfunction in general may be relevant to other age-related disorders, perhaps even 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 from human subjects are divided into aliquots and placed in a U-bottom 96-well microtiter plate. They are then stained with monoclonal antibodies (mAbs). These mAbs, anti-CD8 and anti-KLRG1 or anti-CD103, recognize cell surface markers for a subpopulation of aged T cells. The cells are then stained with pHLA multimers, which recognize antigen-specific receptors on T cells. After incubation, the cells are washed to remove unbound reagents and analyzed using flow cytometry. The percentage of cells that fall within an electronic gate defined by a control strain is used to determine the percentage of PBMCs that bind tumor antigen tetramers.

[0101] Specific Requirements Sterilize all reagents and instruments before use. Maintaining sterility during the procedure is not required but is recommended. Sterilized instruments are to be opened and used only in a sterile hood after they arrive in their labeled manufacturer's packaging. Instruments may be sterilized in autoclavable sterilization pouches (Fisher catalog number 91015 as a suggested supplier). All processing must be performed in a laminar flow hood approved for processing human cells. Universal precautions for the manipulation of human tissue are required.

[0102] Materials / reagents Pipettes (sterilized): P20 Eppendorf (VWR pipette, supplied by Calibrite INC) and P200 Eppendorf (VWR pipette, supplied by Calibrite INC) Pipette tips (sterile, with filter): ART 20 μl nuclease / pyrogen-free tips (Fisher, 2149P) and ART 200 μl nuclease / pyrogen-free tips (Fisher, 2069) Sterile Dulbecco's Phosphate Buffered Saline (PBS) (Invitrogen, 14040-133) Sterile FBS (Gemini Bio. Products, 100-106) Sterile 2% FBS and 98% PBS solution Human anti-CD8 (Pharmigen, unknown number, supplied by T-Neuro) Human anti-KLRG1 (Pharmigen, unknown number, supplied by T-Neuro) Human anti-CD103 (Pharmigen, unknown number, supplied by T-Neuro) Human HLA tumor antigen tetramer, pentamer, or dextramer (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 Sterile U-bottom 96-well microtiter plate (Costar, 3595) Sterile FACS tubes Crushed ice container with lid A clean 1 liter container of bleach Bleach

[0103] Equipment A refrigerated centrifuge capable of spinning at 1400 g 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 a CRO, schedule your FACS run in advance with your facility's operator. Quality Control (Internal): Record results in appropriate laboratory logs and notebooks, including observations and, if GLP is adhered to, signatures by witnesses who did not perform the procedure. The procedure form and checklist must be completed in full and signed by the laboratory director (Quality Assurance Officer) on the same day. Obtain a printout of the FACScan flow cytometer settings from the FACS operator on the day of the FACS run, or obtain the .fcs file for processing and analysis by a responsible party.

[0104] method FACS staining of cell surface markers 1. Using a P200 pipette wiped with 70% ethanol, dispense 2.5 x 10 tetramers in PBS / 2% FBS into each of four wells of a U-bottom 96-well microtiter plate per tetramer to be analyzed. 5 Cells (up to 1 × 10 6 Transfer 100 cells (equivalent number of cells in all wells) of PBMCs or whole blood (e.g. 2 tetramers = 4 wells. If only anti-KLRG1 antibody staining is planned, then there will be just 2 wells per sample). Note: The following procedures do not need to be performed inside a clinical laboratory or laminar flow hood. 2. Centrifuge the plate at 1400 rpm for 5 minutes using the centrifuge located in clinical laboratory D2095. 3. Discard the supernatant by flicking into a clean plastic waste container containing approximately 250-500 ml of bleach, then place the plate on clean paper towels to dry. 4. Transfer the 96-well plate containing cells and antibodies to an ice container packed with ice. 5. Using a clean P200 wiped with 70% ethanol and a sterile pipette tip, resuspend the cells in the wells with 50 μl of the following antibody cocktail: a) 5 μl of anti-CD8 APC + 5 μl anti-CD8103 FITC + 35 μl PBS / 2% FBS; or replace with antibody for other markers b) 5 μl of anti-CD8 APC + 5 μl anti-KLRG1 FITC + 35 μl PBS / 2% FBS Include the same two cocktails for each HLA tumor antigen tetramer being analyzed (e.g., 2 tetramers + up to 2 lines = up to 4 wells total). Incubate cells on ice in the dark for 30 minutes (cover and label the U-bottom microtiter plate to prevent anyone from tipping it over). NOTE: Use a clean pipette tip for each different antibody cocktail dose. 6. Using a clean P200 wiped with 70% ethanol and a sterile pipette tip, add 200 μl of PBS / 2% FBS to each well. Use a clean tip for each well to avoid contamination. 7. Centrifuge the plate at 1400 rpm for 5 minutes with the brake on high. 8. Discard the supernatant by flicking into a clean plastic waste container containing approximately 250-500 ml of bleach, then place the plate on clean paper towels to dry. 9. Using a clean P200 wiped with 70% ethanol and a sterile pipette tip, resuspend the pre-incubated wells with each of the three antibody cocktails and each HLA tumor antigen tetramer solution (one well per tetramer to be analyzed; two tetramers = two wells 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 cells in the dark for 30 minutes at room temperature (exactly 25°C) (cover the plate and label it so no one can tip it over). 11. Using a clean P200 wiped with 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. Centrifuge the 96-well plate at 1400 rpm for 5 minutes with the brake on high using a centrifuge located in the clinical laboratory (D2095). 13. Discard the supernatant by flicking into a clean plastic waste container containing approximately 250-500 ml of bleach, then place the plate on clean paper towels to dry. 14. Using a clean P200 wiped with 70% ethanol and a sterile pipette tip, resuspend the cells in each well with 150 μl of ice-cold PBS / 2% FBS. Resuspend by trituration. Use a clean tip for each well to avoid contamination. 15. Using a clean P200 wiped with 70% ethanol and a sterile pipette tip, transfer the cells into a FACS tube for analysis. 16. Cell Selection Process for Delayed Analysis (Note: Process for stained PBMCs only, not whole blood) 16.1. Using a clean P200 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 pipette up and down in the tube to ensure that the cells and formaldehyde are well mixed. 17. Take the tubes along with the FACS analysis request form to D4029 on the 4th floor. 18. Acquire the results, save the results in the laboratory notebook assigned for FACS analysis, and download the raw.fcs file for subsequent analysis. Reporting the results 1. Within 24 hours of receiving FACS results from the operator, all data will be reviewed and approved by the colleague responsible for performing this assay and the Principal Investigator. 2. Data analyzed are derived from a minimum of 50,000 acquisition events (preferably >250,000) and are presented as the percentage of cells within the viable lymphocyte gate as determined by forward and side light scatter. 3. Minimal staining for both pHLA-empty (>0.7% of cells in the lymphocyte gate) and anti-CD8 (>3.0% of cells in the lymphocyte gate) must be achieved to be eligible for subsequent analysis. 4. The procedure form must be completed in full (including the check mark for the instrument usage record registration) and signed by the laboratory director on the same day. Technical Notes 1. Use only sterile reagents and supplies. 2. Work only in a designated hood. Switch on the hood 10 minutes before use. 3. Wipe the hood clean with 70% ethanol before use. 4. Do not return a pipette used in a flask back into the reagent. 5. Change gloves every time you take your hands out of the hood. 6. Always wear a lab coat, gloves, and protective sleeves 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 above in the detailed description. These descriptions directly describe the embodiments, and it is understood that those skilled in the art may conceive modifications and / or variations to these specific embodiments shown and described herein. Any such modifications or variations that fall within the scope of this description are intended to be included within these embodiments as well. Unless otherwise specified, the inventors intend the words and phrases in this specification and claims to be used in the ordinary and customary manner to those of ordinary skill in the art.

[0106] The foregoing description of various embodiments of the invention known to applicant at the time of filing this application has been presented for purposes of illustration and description. This description is not intended to be exhaustive or to limit the invention to the precise form disclosed, as many modifications and variations are possible in light of the above teachings. The described embodiments serve to illustrate the principles of the invention and its practical application, and also to enable others skilled in the art to utilize the invention in various embodiments and with various modifications to suit the particular uses contemplated. Therefore, it is not intended that the invention be limited to the particular embodiments disclosed for carrying out the invention.

[0107] While particular embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that, based on the teachings herein, changes and modifications can be made without departing from the present invention and its broader aspects, and therefore, the appended claims are to be included within their scope, with all such changes and modifications falling within the true spirit and scope of the present invention. Generally, it will be understood by those skilled in the art that the terms used herein generally refer to "open system" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including but not limited to," etc.).

[0108] As used herein, the terms "comprising" or "comprises" are used when referring to compositions, methods, and individual components thereof that are useful for an embodiment, yet are open to including unspecified elements, whether or not useful. Generally, it will be understood by those skilled in the art that the terms used herein generally refer to "open system" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including but not limited to," etc.). While the open term "comprising," as synonymous with terms such as including, containing, or having, is used herein to describe and claim the present invention, the invention or embodiments of the invention can alternatively be described using alternative terms such as "consisting of" or "consisting essentially of." <Additional Notes> Aspects of the present invention include the following. <Section 1> 1. A method for treating, inhibiting, reducing the severity of, or promoting prevention of age-related cognitive decline, mild cognitive impairment, pathological neurodegeneration, or a combination thereof in a subject in need thereof, comprising: The method comprises administering to the subject a therapeutically effective amount of one or more of a cluster of differentiation (CD103) inhibitor, a perforin 1 inhibitor, and an interferon gamma (IFNγ) inhibitor. <Section 2> the CD103 inhibitor is administered; The CD103 inhibitor is an anti-CD103 antibody, and the clone Ber-ACT 8The method according to item 1, comprising administering to a subject a PE anti-human CD103 antibody derived from the antibody of interest, 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; Item 1. The method according to item 1, wherein the perforin 1 inhibitor comprises a diarylthiophene or GSK2126458. <Section 4> the IFNγ inhibitor is administered; Item 10. The method of claim 1, wherein the IFNγ inhibitor comprises mesopram or rocaglamide. <Section 5> Item 10. The method of item 1, wherein the perforin 1 inhibitor and the IFNγ inhibitor are administered. <Section 6> The method of paragraph 1, wherein the CD103 inhibitor, the perforin 1 inhibitor, and the IFNγ inhibitor are administered. <Section 7> The one or more inhibitors are selected from the group consisting of CD8+ resident memory T cells (T RM ) or effector CD8+ T cells derived from said cells, and RM Alternatively, the method according to Item 1, wherein the binding or response of the effector CD8+ T cells to an amyloid precursor protein (APP) peptide is inhibited. <Section 8> Item 8. The method according to Item 7, wherein inhibiting binding to or response to an APP peptide comprises inhibiting binding to or response to the APP peptide of SEQ ID NO: 8 in the brain. <Section 9> Item 10. The method of item 1, wherein the subject is a human aged 50 or older, 55 or older, 60 or older, 65 or older, or 70 or older. <Section 10> the activity of the effector CD8+ T cells is reduced compared to before administration of the one or more inhibitors or compared to a control subject not administered the one or more inhibitors, and / or the activity of the CD8+ T cells is reduced compared to before administration of the one or more inhibitors or compared to a control subject not administered the one or more inhibitors.RM Item 8. The method according to Item 7, wherein the migration of from the peripheral system to the brain is reduced. <Section 11> 2. The method of paragraph 1, wherein the CD103 inhibitor, the perforin 1 inhibitor, and the interferon gamma (IFNγ) inhibitor independently comprise an antibody, an antigen-binding fragment of an antibody, a small molecule, or a nucleic acid. <Section 12> Item 10. The method of claim 1, wherein the pathological neurodegeneration comprises one or more of multiple sclerosis, Parkinson's disease, and Alzheimer's disease. <Section 13> Item 1. The method according to Item 1, wherein the age-related cognitive decline or mild cognitive impairment has one or more symptoms of short-term or long-term memory loss, decreased ability to maintain concentration, and decreased problem-solving ability. <Section 14> the subject is a human subject; further comprising identifying said human subject as susceptible to or suffering from pathological neurodegeneration prior to said administering; CD103+ resident memory CD8+ T cells (CD8+ T) in the blood of human subjects RM ) is increased compared to values ​​obtained from the same human subject at a younger age without symptoms of age-related cognitive decline, or compared to values ​​obtained from a healthy human subject or a pool of healthy human subjects without symptoms of age-related cognitive decline, the pathological neurodegeneration comprises Parkinson's disease, multiple sclerosis, or Alzheimer's disease; Item 1. The method according to Item 1, wherein the age-related cognitive decline has one or more symptoms of loss of short-term or long-term memory, a decrease in ability to maintain concentration, and a decrease in problem-solving ability. <Section 15> The step of detecting CD103+CD8+T RM 15. The method of claim 14, further comprising detecting elevated CD8A and CD44 levels in the cells. <Section 16> 15. The method of paragraph 14, wherein the human subject is 65 years of age or older. <Section 17> 1. A method of treating, inhibiting, reducing the severity of, or promoting prevention of age-related cognitive decline or pathological neurodegeneration, including multiple sclerosis, Parkinson's disease, or Alzheimer's disease, in a subject in need thereof, comprising: administering a therapeutically effective amount of a vaccine to said subject; The vaccine comprises 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 method. <Section 18> Item 18. The method of Item 17, wherein the subject is a human aged 40 years or older, 50 years or older, 60 years or older, or 70 years or older. <Section 19> 1. A method for identifying a human subject susceptible to or suffering from age-related cognitive decline or pathological neurodegeneration, comprising: The present invention relates to the detection of CD103+ resident memory CD8+ T cells (CD8+ T cells) in blood samples obtained from human subjects with one or more of the following symptoms: loss of short-term or long-term memory, impaired ability to maintain concentration, and impaired problem-solving ability. RM detecting an increased presence of The method. <Section 20> CD103+CD8+T RM 20. The method of claim 19, wherein the increased presence of is compared to a value obtained from a single healthy human subject or a pool of multiple healthy human subjects who do not have any of the one or more symptoms.

Claims

1. 1. A pharmaceutical composition for treating, inhibiting, reducing the severity of, or promoting prevention of pathological neurodegeneration in a subject in need thereof, comprising: the pathological neurodegeneration is Alzheimer's disease, a therapeutically effective amount of a cluster of differentiation (CD103) inhibitor, wherein the CD103 inhibitor is selected from the group consisting of 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 2G5.1 antibody, OX-62, a humanized OX-62 antibody, an anti-mouse CD103 monoclonal antibody derived from clone 2E7, a humanized 2E7 antibody, and paxillin; The CD103 inhibitor inhibits CD8+ resident memory T cells (T ＲＭ ) or effector CD8+ T cells derived from said cells, and ＲＭ or inhibiting the binding or response of said effector CD8+ T cells to amyloid precursor protein (APP) peptides. The pharmaceutical composition.

2. 2. The pharmaceutical composition of claim 1, further comprising a perforin 1 inhibitor and an IFNγ inhibitor, wherein the perforin 1 inhibitor is selected from the group consisting of diarylthiophenes and GSK2126458, and the IFNγ inhibitor is selected from the group consisting of mesopram and rocaglamide.

3. 10. The pharmaceutical composition of claim 1, wherein the subject is a human aged 50 years or older, 55 years or older, 60 years or older, 65 years or older, or 70 years or older.

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