BIOMARKERS AND THERAPEUTIC TARGETS FOR NEURODEGENERATIVE DISEASES INVOLVING K311 -ACETYLATED TAU

CROSS REFERENCE TO OTHER APPLICATIONS

[0001 ] This application claims the benefit of U.S. Provisional Application No. 63/296,042, filed January 3, 2022, the contents of which are hereby incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

[0002] A sequence listing is provided herewith as a sequence listing xml, “S21 -422_STAN- 1916WO_Seqlist” created on December 23, 2022 and having a size of 4 290,456 Bytes. The contents of the sequence listing xml are incorporated by reference herein in their entirety.

BACKGROUND

[0003] Alzheimer’s (AD) and Parkinson’s (PD) diseases, and Amyotrophic Lateral Sclerosis (ALS) and other neurodegenerative diseases, are responsible for considerable morbidity and mortality. With incidence rising with aging, these also represent a growing societal problem. Pathophysiology of these diseases involves accumulation of Tau (neurofibrillary tangles) and Amyloid-|3-rich (amyloid plaques) aggregates in AD, alpha-synuclein-rich aggregates (Lewy body) in PD and tar DNA-binding protein-43 (TP-43) rich aggregates in ALD. Innate immune responses and microglial involvement are also involved.

[0004] More recently, a role for adaptive immunity has been outlined through genetic and immunological studies. Indeed, PD, AD, ALS and other neurodegenerative diseases have genome-wide association study (GWAS) signals in the Human Leukocyte Antigen (HLA, also called the Major Histocompatibility Complex, MHC) region. Although initially attributed to HLA- DRA, DRB5 or DRB1 *15:01 , recent studies demonstrated that the PD HLA signal marks HLA- DRB1 *04. The AD and ALS HLA signals were less well characterized. Additionally, a comparison of healthy controls and individuals with neurodegenerative disorders showed that a complex polyclonal B and T cell response develops against alpha-synuclein, tau, and TDP- 43, proteins involved in pathological aggregates. Whether or not these responses are bystander, contributes to, or protects against neurodegeneration is unknown.

[0005] There are very limited pharmacologic agents to treat or prevent neurodegenerative diseases involving Tau, such as AD, PD, or ALS, and treatment therefore presents an unmet need.

SUMMARY [0006] The present disclosure provides compositions and methods for treating a mammalian subject with a neurodegenerative disease involving Tau including, for example, Alzheimer’s disease (AD), Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Compositions may comprise an anti-Tau agent and a pharmaceutically acceptable excipient.

[0007] As described in the present disclosure, a genome-wide association study (GWAS) data of HLA associations in individuals with AD or PD was analyzed to identify protective effects of HLA-DRB1 *04 subtypes. This was found to be primarily associated with decreased phospho- Tau (or total-Tau) but not strongly Amyloid-|342 levels in the cerebrospinal fluid and with reduced neurofibrillary tangles, but not amyloid plaques, in post-mortem brains. Specific DRB1 *04 subtypes strongly bound aggregation-prone R3-repeat Tau PHF6 sequences but only when K311 was acetylated, a modification central to aggregation and particularly common in AD brains.

[0008] In the methods of the disclosure, an effective dose of an anti-Tau agent acting on acetyl-PHF6 or structural equivalent is administered to an individual having, or at risk to have, a neurodegenerative disease involving Tau directly or as a modulator of pathology, in a dose effective to stabilize, reduce or prevent clinical symptoms of the disease. A variety of neurodegenerative diseases involving Tau may be treated by practicing the methods, including AD, PD, ALS, Pick’s disease, Lewy Body Dementia, progressive supranuclear palsy, corticobasal degeneration, Frontotemporal dementias, chronic traumatic encephalopathy (GTE) or post head trauma treatment aiming at preventing tau aggregation in these patients, argyrophilic grain disease, globular glial Tauopathies, Vacuolar Tauopathy, Lytico-bodig disease, postencephalitic parkinsonism, subacute sclerosing panencephalitis, primary age- related tauopathy, which includes chronic traumatic encephalopathy (GTE) and aging-related tau astrogliopathy.

[0009] The effects of anti-Tau agents on neurodegenerative diseases involving Tau such as AD, PD, or ALS can include a range of outcomes, which are optionally monitored following treatment. For instance, outcomes may include a reduction in symptoms associated with AD such as cognitive impairment which includes attention and concentration, the ability to learn complex tasks and concepts, memory, information processing, visuospatial function, the ability to produce and understanding language, the ability to solve problems and make decisions, and the ability to perform executive functions, etc. Methods of treatment disclosed herein may provide for a reduction in symptoms associated with PD such as unilateral resting tremor, decreased movement, rigidity, postural instability, or dementia. Methods of treatment disclosed herein may provide for a reduction in symptoms related to ALS such as reduced muscle weakness, muscle atrophy, fasciculations, emotional lability, or respiratory muscle weakness relative to no treatment. Methods for monitoring treatment responses may comprise, for example, measuring tau fragment biomarkers or aggregates, and tau brain imaging studies.

[0010] In some embodiments an anti-Tau agent is an immunogenic peptide comprising a post- translationally modified (PTM) Tau PHF6-like sequence, e.g. (SEQ ID NOT) 3 ⁰⁶ VQIVY(acetylK)PVDLSK ³¹⁷ (PHF6, acetylated at K311 ) or an homologous sequence, including without limitation (SEQ ID NO:8) ²⁷⁵ VQIIN(acetylK)KLDLSNV ²⁸⁷ (PHF6*, acetylated at K280), (SEQ ID NO:12) SVQIVY(acetyl-K)PVDLS; (SEQ ID NO:113) SVQIVYKPVDLS[acetyl]KVT; (SEQ ID NO:114) SVQIVY[Acetyl]KPVDLSKVT; (SEQ ID NQ:107) SVQIVYKPVDSK[Acetyl]VT; or similar simple amino acid substitutions of PHF6, notably those using Q, K methyl, K dimethyl instead of K acetyl or any change that will allow binding to DRB1 *04. An immunogenic peptide is administered in a formulation to elicit a protective immune response specific for the PTM peptide, which immune response decreases Tau mediated aggregation. This is directly demonstrated by the demonstration herein that in individuals without neurodegeneration, a DRB1 *04 restricted immune response against tau can be present, whereas in DRB1 *04 controls, a regulatory, anti-inflammatory T cell response (thus non-protective) can be present. One aspect of the present disclosure is promotion of a protective immune response, as initially identified in control subjects with DRB1 *04.

[0011] Treatments relating to an immunogenic response, i.e. vaccination with an effective dose of a PTM Tau peptide such as SEQ ID NOT, 8, 12, 107, 113, 114, or homologous peptide sequence thereof are particularly beneficial for individuals carrying at least one HLA-DRB1 *04 allele. The individual may have any subtype of HLA-DRB1 *04, including DRB1 *04:01 , DRB1 *04:02, DRB1 *04:03, DRB1 *04:04, DRB1 *04:05, DRB1 *04:06, DRB1 *04:07, DRB1 *04:08, DRB1 *04:09, DRB1 *04:10, DRB1 *04:11 , DRB1 *04:12, DRB1 *04:13 or the similar DRB4*01 :01 , DRB4*01 :03 alleles of DRB4, a gene different from DRB1. Individuals comprising an HLA-DRB1 *07 or HLA-DRB1 *09, which carry DRB4*01 :01 or DRB4*01 :03 genes homologous to DRB1 *04 may also receive benefit from vaccine treatments.

[0012] In some embodiments, a method is provided for treatment of a human subject for AD, the method comprising administering an effective dose of an immunogenic PTM Tau peptide such as SEQ ID NOT, 8, 12, 107, 113, 114, or homologous peptide sequence thereof. In some embodiments, a method is provided for treatment of a human subject for PD, the method comprising administering an effective dose of an immunogenic PTM Tau peptide such as SEQ ID NOT, 8, 12, 107, 113, 114, or homologous peptide sequence thereof. In some embodiments, a method is provided for treatment of a human subject for ALS, the method comprising administering an effective dose of an immunogenic PTM Tau peptide such as SEQ ID NOT, 8, 12, 107, 113, 114, or homologous peptide sequence thereof. In some embodiments, a method is provided for treatment of a human subject with head trauma or other tauopathies, the method comprising administering an effective dose of an immunogenic PTM Tau peptide such as SEQ ID N0:7, 8, 12, 107, 113, 114, or homologous peptide sequence thereof.

[0013] In other embodiments, an anti tau agent may be a drug or small molecule that binds the HLA complex when it interacts with a PHF6-like sequence, or a PHF6 like modified sequence such that it allows the interaction of HLA subtypes other than a subtype of HLA- DRB1 *04 with the PHF6-like sequence to occur, allowing an immune response against PHF6- like sequences to be mounted in individuals without DRB1 *04.

[0014] In other embodiments an anti-Tau agent is an agent that specifically binds to and inhibits the aggregation of acetylated Tau protein by binding Tau that is acetylated at K311 (inside PHF6 sequence) or K280 (inside the PH6* sequence, homologous in sequence to PHF6), or degrades or disaggregates neurofibrillary tangles (NFTs) comprising K311 and K280 acetylated Tau proteins. Such anti-Tau agents specifically recognize the PTM epitope of Tau that is acetylated at K311 or homologous K280, which agents include, without limitation, antibodies, antibody fragments, single antigen binding regions (ABR), chimeric antibodies, chimeric antigen receptor (CAR) T cells, etc. Examples of TCR antigen-binding regions (ABR) are provided, for example, in Table 2 herein, and may comprise a sequence with 1 , 2, 3, 4, 5, 6, 7, 8, 9, or with up to 10 amino acid substitutions within the CDR3alpha and beta sequences combined, such that it can maintain HLA-peptide specificity (see for example Luo et al. Proc Natl Acad Sci U S A. 2022 Aug 9;119(32):e2205797119, which shows up to 10 substitutions but not more can maintain binding).

[0015] Any individual may be treated with an agent that specifically binds to and inhibits the aggregation of the acetylated Tau protein by interfering with PHF6 or a related sequence, regardless of their HLA-DRB1 type. For instance, an individual may have an HLA-DRB1 *01 , HLA-DRB1 *03, HLA-DRB1 *04, HLA-DRB1 *07, HLA-DRB1 *08, HLA-DRB1 *09, HLA- DRB1 *10, HLA-DRB1*11 , HLA-DRB1 2, HLA-DRB1 3, HLA-DRB1 4, HLA-DRB1 5, or an HLA-DRB1 *16 and a modified PHF6 sequence that is designed to bind these subtypes. Our current data found that no other HLA class II subtype beside DRB1 *04 or DRB4*01 can bind PHF6 or acetyl PHF6.

[0016] In some embodiments, a method is provided for treatment of a human subject for AD, the method comprising administering an effective dose of a targeted anti-Tau agent that specifically binds to and inhibits the aggregation of acetylated Tau protein by binding Tau that is acetylated at K311 or K280. In some embodiments, a method is provided for treatment of a human subject for PD, the method comprising administering an effective dose of a targeted anti-Tau agent that specifically binds to and inhibits the aggregation of acetylated Tau protein by binding Tau that is acetylated at K311 or K280. In some embodiments, a method is provided for treatment of a human subject for ALS or after head trauma, the method comprising administering an effective dose of a targeted anti-Tau agent that specifically binds to and inhibits the aggregation of Tau by binding Tau that is acetylated at K311 or K280.

[0017] In various aspects and embodiments, the methods may include administering to an individual suffering from neurodegenerative diseases such as AD, PD or ALS an effective dose of an anti-Tau agent, where the treatment reduces or stabilizes clinical symptoms of the disease. In one embodiment, the individual is a human. In some embodiments the anti-Tau agent is combined with a secondary therapeutic agent, including without limitation carbidopa/levodopa, anticholinergic drugs (e.g. pramipexole, ropinirole), MAO-B inhibitors (selegiline, rasagiline), amantadine, dopamine agonists, (e.g., pramipexole, ropinirole, rotigotine), catechol O-methyltransferase (COMT) inhibitors (eg entacapone, opicapone, tolcapone), aducanumab, donepezil, rivastigmine, memantine, manufactured combination of memantine and donepezil, galantamine, riluzole, baclofen, quinine, phenytoin, glycopyrrolate, amitriptyline, benztropine, trihexyphenidyl, transdermal hyoscine, atropine, fluvoxamine, dextromethorphan, quinidine, gabapentin, etc. In some embodiments, more than one anti-Tau agent may be administered to an individual. In other embodiments the anti-Tau agent may be administered to an individual together with other therapies targeting other pathological aggregates (anti-beta-amyloid, anti-alpha-synuclein, anti-TP43) or other parts of Tau that do not contain PHF6 like sequences.

[0018] The effective dose of each drug in a combination therapy may be lower than the effective dose of the same drug in a monotherapy. In some embodiments the combined therapies are administered concurrently. In some embodiments the two therapies are phased, for example where one compound is initially provided as a single agent, e.g. as maintenance, and where the second compound is administered during a relapse, for example at or following the initiation of a relapse, at the peak of relapse, etc.

[0019] Another aspect of the methods includes a method of generating and screening candidate anti-Tau agents. Anti-Tau agents may be generated using any suitable method. Suitable methods for the generation and screening of anti-Tau agents, include without limitation, immunization of dromedaries, immunization of rabbits, immunization of goats, immunization of sheep, immunization of horses, immunization of chickens, immunization of mice, immunization of rats, immunization of guinea pigs, immunization of camels, immunization of alpacas, immunization of sharks, yeast surface display, etc. When using immunization or yeast surface display, immunization and yeast display are conducted using an epitope or peptide of acetylated Tau, i.e. Tau acetylated at K311 and/or K280. Antibodies generated from experimental animals may be further modified for use in humans thereby generating humanized antibodies or fragments or peptides modified from these sequences, are to obtain the same results, for example by replacing acetyl-K by Glutamine (Q) or methyl- K, may be used to generate anti-Tau agents. [0020] Screening agents for monitoring and detection of T cells involved in anti-Tau immune responses are provided, including MHC tetramers/multimers with DRB1 *04:01 , DRB1 *04:02, DRB1 *04:03, DRB1 *04:04, DRB1 *04:05, DRB1 *04:06, DRB1 *04:07, DRB1 *04:08,

DRB1 *04:09, DRB1 *04:10, DRB1 *04:11 , DRB1 *04:12, DRB1 *04:13, DRB4*01 :01 ,

DRB4*01 :03, or other subtypes more homologous to DRB1 *04:04 than to any other natural non DRB1 -04 allele in combination with a Tau peptide, e.g. any of SEQ ID NO:66-109. DRB1 *04:01 and DRB1 *04:04 tetramers presenting a PTM Tau peptide comprising acetylated K311 or K280 (or equivalent sequences) are of particular interest. The tetramers are useful in determining the presence of reactive T cells to Tau aggregation motifs. In some embodiments a method is provided for screening T cells from an individual for the presence of a protective response, the method comprising binding a T cell population to an MHC tetramer presenting a PTM Tau peptide comprising acetylated K311 or K280, and determining the number and/or frequency of binding T cells in the population. The number and/or frequency may be compared to a control population. The individual may be treated accordingly, where increased numbers of protective T cells indicate less risk of developing neurodegenerative diseases such as AD, PD or ALS. The T cells carrying this specificity may also be characterized for gene content, subpopulation type such as regulatory, cytotoxic or effector/memory phenotypes, where Treg cells are associated with a non-protective response.

[0021] In one embodiment, a therapeutic modality is provided, comprising a physiological manipulation to transform a regulatory T cell response against PHF6-like sequences to a memory/effector T cell response against PHF6-like sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features can be arbitrarily expanded or reduced.

[0023] FIG. 1 . Colocalization of the HLA locus signal in Alzheimer’s and Parkinson’s diseases in the largest multi-ancestry meta-analyses to date of these two neurodegenerative diseases. Posterior probability of colocalization (PP4) = 0.99. Additionally, colocalization with the HLA locus of ALS is also shown.

[0024] FIG. 2: The pro-aggregation PHF6 region of Tau binds HLA-DRB1 *04 subtypes only when acetylated at K311 . Fifteen-mer peptides (800 pM) encompassing the entirety of all Tau isoforms (schematized in the top panel), overlapping across 11 residues, were screened for HLA-DRB1 *04:01 , HLA-DRB1 *04:04 and HLA-DRB1 *04:05 binding (see methods), with and without common PTMs as reported by Wesseling et al. Four regions (labelled in purple, red, blue, orange) containing strong HLA-DRB1 *04 binders (log displacement <1 -1.4, below 25% of baseline control) were further tested at various concentrations (bottom panel), showing three promising regions where binding was stronger with HLA-DRB1 *04:04/ HLA- DRB1 *04:01 , intermediary with HLA-DRB1 *04:03 and absent or weak with HLA-DRB1 *04:05 and HLA-DRB1 *04:06, a pattern similar to GWAS risk (Table 1). Among these regions, PHF6 3 ⁰⁶ VQIVY(acetylK)PVDLSK ³¹⁷ strongly binds HLA-DRB1 *04:01 , HLA-DRB1 *04:03 and HLA- DRB1 *04:04 only when post-translationally modified at K311 . This segment is well known to seed Tau aggregation, and this process is known to be modulated in the presence of acetyl K311 .

[0025] FIG. 3. Immune clearance of acetylated PHF6 Tau sequences reduces neurodegeneration in AD and PD. Pathological Tau seeds, soluble Tau fragments, or misfolded Tau present in the extracellular space are taken up and phagocytosed by activated microglia where it is processed. In addition to autophagy, resulting Tau peptide fragments, notably acetylated lysine (K-ac) 311 PHF6 are bound to HLA-DRB1 *04:01 or HLA- DRB1 *04:04 and the resulting HLA-peptide complexes presented by microglial cells (or other antigen presenting cells) to CD4 ⁺ T helper cells. CD4 ⁺ T cells trigger beneficial downstream immune responses perhaps involving CD8 ⁺ T and antibody producing B cells. These responses limit propagation of misfolded Tau and reduce neuropathology, also explaining reduced CSF Tau in HLA-DRB1 *04 individuals.

[0026] FIG. 4. Haplotypes harboring key DRB1 *04 subtypes and/or DQB1 *03:02. Effect sizes highlighted in red were nominally significant (p < 0.05).

[0027] FIG. 5. Locuszoom plots of the local-HLA GWAS in multi-ancestry AD, PD, and ALS analyses. The lead variants in the AD and PD GWAS are missing in the summary statistics of the latest ALS GWAS, consequently the colocalization is imperfect.

[0028] FIG. 6. Linkage disequilibrium of the lead SNP with HLA-DRB1 amino acids and DRB1 *04 allele subtypes.

[0029] FIG. 7. HLA-DR4 binding motifs and PHF6 sensitivity. Residues of PHF6 in their physiological or PHF6-K311 forms are shown and their association with the binding motifs of (a) HLA-DRB1*04 04, (b) HLA-DRB1*04 0~\ and (c) HLA-DRB1*04 05. Motifs are adapted from Scally et al., 2013 (/-/L4-DF?B7*04:01 and HLA-DRB1*04 04) and Ting et al., 2018 (HLA- DRB1*0405).

[0030] FIG. 8. HLA binding predictions for PHF6. Predictions were made using NetMHGIIPan 4.0 (left) using Mixed MHO pred 2 Server (right). Cmap indicates a percentile rank generated by comparing the peptide's score against the scores of five million random 15 mers selected from SWISSPROT database (best score = 0%; worst score = 100%). 15mer sequences incorporating 15mer sequences incorporating PHF6 in its unacetylated (bottom) or K311 mimic acetylated (top) form. Annotations within heat map show the position of the lysines of interest (K311 for PHF6, see highlighted sequences) within the 9mer core that is predicted to bind to the HLA molecule. 9mer cores excluding the lysine of interest were not predicted for binding and left blank. Note prediction of PHF6 binding to HLA-DRB1*04 subtypes associated with neurodegeneration, confirmed by our in vitro experiments and the absence of binding to predicted to other common HLA DR and DQ subtypes. One exception is prediction of binding of the PHF6 sequence to HLA-DRB4*0t alleles for both acetylated and non-acetylated PHF6 sequences. Accessory HLA-DRB4 genes are more weakly expressed than HLA-DRB1. These genes are present not only on HLA-DRB1*04 haplotypes, but also on most HLA-DRB1*07'.(y\ and HLA-DRB1*09 0'\ haplotypes. As HLA-DRB o '.Ot and HLA-DRB1*09 0'\ haplotypes are not associated with AD or PD, HLA-DRB4*<y\ is less likely to be involved in disease predisposition, maybe because of low expression and binding to both acetylated and nonacetylated sequences resulting in central tolerance. Monitoring HLA-DRB4*<y\ -PHF T cell reactivity may be utilized for monitoring patients with unusual response or non-response to the anti-acetylated PHF6 therapy.

[0031] FIG. 9. DRB1 *04 subtypes do not show increased binding to a-synuclein in the presence of common PTMs (SEQ ID NO:118-179) for this protein. Note that DRB1 *04:04 and 04:01 strongly bind to the reference in the region 121 -135 but their binding efficiency decreases in the presence of PTMs pY125 and/or pS129. See methods for details.

[0032] FIG. 10. HLA alleles DRB1 *04:04 and DRB1 *04:01 are associated with a decreased risk of Parkinson’s and Alzheimer’s diseases. Effect sizes are reported as odds ratio (OR), with 95% confidence interval [Cl], and significance (p-value).

[0033] FIG. 11 . rs601945, an equivalent marker of HLA-DRB1 *04, is associated with reduced Tau in CSF and postmortem brain tissues, with protection against Lewy-body pathology, but association with increased A|342 and decreased neuritic plaques density is weak. p-Tau: phosphorylated Tau, t-Tau: total Tau, N: number of individuals, MAF: minor allele frequency, OR: odds ratio, |3: parameter estimate, Cl: confidence interval. Braak: Tau Braak staging, Neur: Neuritic plaques density.

[0034] FIG. 12. Number of individuals per disease and ancestry group included in the metaanalyses.

[0035] FIG. 13. DRB1 *04 alleles are associated with reduced Tau and neurofibrillary tangles but not with Amyloid-|3 or neuritic plaques, when testing their association with Alzheimer’s disease neuropathology and cerebrospinal fluid biomarkers. p-Tau: phosphorylated Tau, t- Tau: total Tau, N: number of individuals, MAF: minor allele frequency, OR: odds ratio, |3: parameter estimate, Cl: confidence interval. Braak: Tau Braak staging, Neur: Neuritic plaques density. [0036] FIG. 14. Queried US based cohorts’ part of the Alzheimer’s disease ADSP and ADGC analyses.

[0037] FIG. 15. Demographics of the cohorts queried among the ADSP and ADGC in-house analyses in Alzheimer’s disease. AFR: African, AMR: American (central and south; admixed), EAS: East Asian, SAS South Asian, EUR: European, otherwise ADMIX: admixed of these super ancestry categories.

[0038] FIG. 16. Demographics by ancestry of ADSP and ADGC individuals included in the analyses. AAD: age-at death, AAL: age-at-last-exam, AAE: age-at-exam, AAO: age-at-onset.

[0039] FIG. 17. Demographics by ancestry of ROSMAP and ADGC individuals included in the neuropathology analyses. AAD: age-at death, AAL: age-at-last-exam, AAE: age-at-exam, AAO: age-at-onset.

[0040] FIG. 18. Demographics by ancestry of ROSMAP and ADGC individuals included in the dual-pathology analyses. AAD: age-at death. AD-LB-: no AD and LB pathology, AD+LB-: only AD pathology without LB pathology, AD-LB+: only LB pathology without AD pathology, AD+LB+: dual pathology (AD and LB).

[0041] FIG. 19. AD pathology only, Lewy body (LB) pathology only, and dual pathology (AD and LB) and compared these against controls without AD and LB pathologies.

[0042] FIG. 20. Fluorescence Activated Cell sorting experiments showing presence of CD4 T cells recognizing acetylated form of PHF6 (dots in the upper right quadrant) in individuals with DRB1 *04:01 or DRB1 *04:04 (2 controls and 2 patients with narcolepsy; the fact the subjects have narcolepsy is inconsequential for this experiment and can be considered as an additional control). These experiments used DRB1 *04:01 and DRB1 *04:04 tetramers loaded with K311 acetylated peptide PHF6. The first three columns represent staining with DRB1 *04:01 and DRB1 *04:04 tetramers loaded with tau 5-19, tau 404-418 and tau 421-435 from Figure 2. No cells (dots) are detected in the upper corner with these other sequences, indicating no T cells recognizing these other tau epitopes, unlike with SVQIVY(acetylK)PVDLSKVT (acetyl PHF6). This is likely due to central tolerance to non PTM-modified sequences of Tau.

[0043] FIG. 21 HLA binding predictions for PHF6*, a segment homologous to PHF6. Predictions were made using NetMHGIIPan 4.0 (left) and using Mixed MHO pred 2 Server (right). Cmap indicates a percentile rank generated by comparing the peptide's score against the scores of five million random 15 mers selected from SWISSPROT database (best score = 0%; worst score = 100%). 15mer sequences incorporating 15mer sequences incorporating PHF6* in its unacetylated (bottom) or K2380 mimic acetylated (top) form. Annotations within heat map show the position of the lysines of interest (K280 for PHF6*, see highlighted sequences) within the 9mer core that is predicted to bind to the HLA molecule. 9mer cores excluding the lysine of interest were not predicted for binding and left blank. Note that although acetylated 280K PHF6* is predicted to bind DRB1 *04, it was not found to bind DRB1 *04 in vitro (FIG. 2 and FIG. 22). It is also predicted to bind DRB4*01 :01 or DRB4*01 :03 whether or not it is acetylated at K280, as for PHF6 whether or not it is acetylated at K311 . Based on this and the high homology of PHF6 and PHF6* at the amino acid level, we consider PHF6* a PHF6-like sequence as it may behave as PHF6 when administered in a vaccine or used as a reagent.

[0044] FIG. 22. PHF6* peptides do not bind DRB1 *04 subtypes. Each Tau peptide at different concentrations competed with biotinylated peptide binding to HLA-DRB1*04'y\ , HLA- DRB1*0403, HLA-DRB1*04\04, HLA-DRB1*04:05, and HLA-DRB1*04\06. Tau peptides with fluorescence that was lower than 25% and 25-50% of biotinylated peptide are considered strong (SB) and weak (WB) binders, respectively. None of the peptides bind these subtypes.

[0045] FIGS. 23A-23C. Screening of tau peptides binding DRB1 *04 alleles, (a) the most frequent PTMs tested, (b) Screening for DRB1 *04:04/01/05. Four regions out-competed 50% of bio-GAD were indicated, (c) Dose response of the four regions including two more DRs: DRB1 *04:03 and DRB1 *04:06. pS/T/Y, phosphorylated serine/threonine/tyrosine; mK, methylated lysine; aK, acetylated lysine; SB, strong binder; WB, weak binder.

[0046] FIGS. 24A-24B. Selected tau peptides binding affinity against HLA-DRB4*01 :03, DQA1 *03:01 -DQB1 *03:02 and DQA1*01 :01 ~DQB1 *05:01. (a) Binding of 44 tau DR401/4- binders, SEQ ID NO:66-SEQ ID NO:109, against DRB4*01 :03. Strong and weak binders are highlighted in green and orange, respectively. DR and Bio-GAD are negative and positive controls, respectively, (b) Four regions of tau binding affinity, SEQ ID NO:110-SEQ ID NO:116 against DQA1 *03:01 -DQB1 *03:02 and DQA1 *01 :01 -DQB1 *05:01 .

[0047] FIGS. 25A-25C. T cells reactivity toward acetylated PHF6 tau sequences involvement in neurodegenerative diseases. (A) Example of FACS plots showing T cell population recognizing these sequences when presented by HLA-DRB1 *04:01 and HLA-DRB1 *04:04. (B) T cell abundance in HLA-DRB1 *04:01 versus HLA-DRB1 *04:04 subjects in various disease groups. (C) Significantly higher numbers of cells in HLA-DRB1 *04:04 subjects, and a trend toward lower numbers in patients versus controls. Same subjects with longitude are connected with arrows. Subject ID is shown under each plot panel. AD, Alzheimer’s disease. MCI, mild cognitive impairment. PD, Parkinson’s disease. HC, healthy control. LBD, Lewy body dementia. Y, year.

[0048] FIG. 26. Restricted CD4 ⁺ T cells by multiple tau epitopes in one single staining. PBMCs were cultured with a pool of tau peptides (Tau-449, 536, 527, 446 and 2) for 10 days and stained with a combinatorial panel of tetramers HLA-DRB1 *04:04/01 -cognate tau epitopes. AD, Alzheimer’s disease. MCI, mild cognitive impairment. Subject ID is shown on the right of staining panels. This shows that tau pieces other than acetylated PHF6 bound to DRB1 *04 also recognize some T cells in some subjects. These could be used as controls when studying the DR4-acetylated PHF6 reactivity. [0049] FIG. 27 T cell receptor (TOR) sequencing of tau acetylated K311 PHF6 (K311Ac) restricted CD4 ⁺ T cell. One HLA-DRB1 *04:04 positive AD and one control were stained with tetramer DRB1 *04:04-cognate tau peptide and PHF6 (K311Ac) restricted CD4 ⁺ T cells were sorted into 96-well plate. TCR clones were recovered and enriched. AD, Alzheimer’s disease. HC, healthy control.

[0050] Figure 28. Cluster and phenotyping of tau PHF6 (K311 Ac) restricted CD4 ⁺ T cell. 82 AD and 76 HC cells that passed quality control were cluster using a standard workflow and UMAP. TCR clones were mapped back to single cells. Expression of Treg markers FOXP3 and CTLA4 were compared between AD and HC and between clusters, and CD3E as a control, p-value was shown with age as covariant. AD, Alzheimer’s disease. HC, healthy control. It is shown that the T cells recognizing PHF6 presented by DRB1 *04:04 are almost all regulatory T cells in the Alzheimer patient, and regular memory/effector T cells in the control. This indicates that only the T cells of the healthy individual provide an immune response that will clear PHF6 containing tau fragment. T cells of the AD patient may inhibit this favorable response, explaining why they developed neurodegeneration despite having HLA- DRB1 *04:04, a protective genetic marker.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0051 ] All structural and functional equivalents to the features and method acts of the various embodiments described throughout the disclosure that are known or later come to be known to those of ordinary skill in the art are intended to be encompassed by the features described and claimed herein. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. No claim element is to be construed under the provisions of 35 USC 112, sixth paragraph, unless the element is expressly recited using the phrase "means for" or "step for”.

[0052] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0053] Methods recited herein may be carried out in any order of the recited events which is logically possible, as well as the recited order of events.

[0054] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0055] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0056] As used herein, the term "subject" encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. The term does not denote a particular age or gender.

[0057] As used herein, the terms "treat," "treating" or "treatment," and other grammatical equivalents, include: alleviating, abating or ameliorating one or more symptoms of a disease or condition. In some embodiments, treating is alleviating, abating, or ameliorating one or more symptoms of a neurodegenerative disease involving Tau. In some embodiments, treating is alleviating, abating or ameliorating one or more symptoms of AD. In some embodiments, treating is alleviating, abating or ameliorating one or more symptoms of PD. In others it is reducing a proxy marker of the tau pathology, for example the tau burden measured by imaging, or the amount of tau peptide in body fluids, notably when acetylated or methylated at K311.

[0058] By the terms "effective amount" and "therapeutically effective amount" of a formulation or formulation component is meant a sufficient amount of the formulation or component, alone or in a combination, to provide the desired effect. For example, by "an effective amount" is meant an amount of an anti-tau agent, alone or in a combination, required to treat or prevent a neurodegenerative disease involving Tau in a mammal. Ultimately, the attending physician or veterinarian decides the appropriate amount and dosage regimen.

[0059] The effective dose of a therapeutic composition to be given to a particular patient will depend on a variety of factors, several of which will be different from patient to patient. Utilizing ordinary skill, the competent clinician will be able to optimize the dosage of a particular therapeutic or imaging composition in the course of routine clinical trials. [0060] Tau is a soluble protein that normally binds to microtubules and regulates their dynamic growing and shortening behaviors. In Alzheimer’s disease (AD) and other neurodegenerative diseases, Tau dissociates from microtubules and self-associates to form abnormal fibrillar aggregates. The distribution of these aberrant Tau structures is very well-correlated with neuronal cell death and the clinical progression of neuodegenerative diseases, suggesting an intimate link between aberrant Tau structure and neurodegeneration/dementia. Recent efforts to identify the neurotoxic species of Tau have shifted focus away from mature, fibrillar aggregates toward smaller oligomeric Tau species. Studies with antibodies that selectively recognize Tau oligomers have demonstrated that these species are elevated in AD brains. Cell to cell transmission of oligomeric Tau and other aberrant pre-fibrillar species may underlie the spread of Tau pathology, and these species show promise as potential therapeutic targets. A structural understanding of early Tau aggregates may lead to a deeper understanding of their neurotoxic mechanisms, as well as to the rational design of therapeutic drugs.

[0061] As a result of alternative RNA splicing there are 6 distinct isoforms of Tau that differ from one another depending upon the presence or absence of three inserts encoded by exons 2, 3 and 10 of the Tau gene. There are two important domains in each Tau isoform: the N- terminal projection domain determines the inter-microtubule spacing between bundled microtubules and also mediates interactions of microtubules with plasma membrane. Exons 2 and 3 each encode 29-residue acidic inserts located in the N-terminal projection domain. In contrast, the microtubule binding pseudo-repeat (MTBR) domain contains either three or four imperfect repeats (depending upon the presence or absence of exon 10 encoded sequences) and serves to bind microtubules directly and to regulate their dynamics. This same region of the protein makes up the core of fibrillar T au aggregates, and T au aggregation is accompanied by a regional transition from random coil to /3-sheet structure. Fibrillar Tau aggregates have a cross-jB-structure typical of amyloid fibrils. Finally, the 6 tau isoforms differ from one another not only structurally but also in terms of the relative expression levels and their rates and extents of fibril formation. Genetic evidence demonstrates unequivocally that functional differences must exist among the 6 different tau isoforms.

[0062] The amino acid sequences of the six human tau isoforms include the following: isoform 1 (SEQ ID NO: 1 ) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDE AAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPP APKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPS SAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNI H HKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHG A EIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL.

[0063] Isoform 2 (SEQ ID NO: 2):

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGS

EEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKA K

GADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSP G

SPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKS KI GSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDR VQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLS NVSSTGSIDMVDSPQLATLADEVSASLAKQGL.

[0064] Isoform 3 (SEQ ID NO: 3):

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGS

EEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPS LE

DEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIP AKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVR TPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTS KCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFR ENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAK QGL

[0065] Isoform 4 (SEQ ID NO: 4):

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSL

EDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRI PAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVV RTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQ

SKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLD F KDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSP RHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL.

[0066] Isoform 5 (SEQ ID NO :5):

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGS

EEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKA K

GADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSP G

SPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKS KI

GSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSK VT

SKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKL TF

RENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSAS LA KQGL

[0067] Isoform 6 (SEQ ID NO: 6): MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGS EEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLE DEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIP AKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVR TPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQS KCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFK DRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPR HLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL. When specific amino acid numbers are referenced, the numbering refers to SEQ ID NO: 6 unless specifically indicated otherwise

[0068] The terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction. In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD (dissociation constant) of 10 ^-5 M or less (e.g., 10' 6 M or less, 10' ⁷ M or less, 10' ⁸ M or less, 10' ⁹ M or less, 10' ¹⁰ M or less, 10' ¹¹ M or less, 10' ¹² M or less). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), e.g. as used by Biacore systems. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g. at 25°C.

[0069] MHO Proteins. Major histocompatibility complex proteins (also called human leukocyte antigens, HLA, or the H2 locus in the mouse) are protein molecules expressed on the surface of cells that confer a unique antigenic identity to these cells. MHC/HLA antigens are target molecules that are recognized by T-cells and natural killer (NK) cells as being derived from the same source of hematopoietic reconstituting stem cells as the immune effector cells ("self") or as being derived from another source of hematopoietic reconstituting cells ("non-self"). Two main classes of HLA antigens are recognized: HLA class I and HLA class II.

[0070] The MHO proteins of interest are the human HLA proteins. Included in the HLA proteins are the class II subunits HLA-DPA1 , HLA-DPB1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA and HLA- DRB, and the class I proteins HLA-A, HLA-B, HLA-C, and [32-microglobulin.

[0071 ] HLA-DRB1 *04 is of particular interest which can be further divided into DRB1 *04:01 , DRB1 *04:02, DRB1 *04:03, DRB1 *04:04, DRB1 *04:05, DRB1 *04:06, DRB1 *04:07, DRB1 *04:08, DRB1 *04:09, DRB1 *04:10, DRB1 *04:1 1 , DRB1 *04:12 and DRB1 *04:13. The DRB4*01 :01 and DRB4*01 :03 genes are accessory DRB genes similar to DRB1 *04 that are carried by individuals with DRB1 *04, DRB1 *07 and DRB1 *09. These have similar but not identical binding properties than DRB1 *04. [0072] An “allele” is one of the different nucleic acid sequences of a gene at a particular locus on a chromosome. One or more genetic differences can constitute an allele. An important aspect of the HLA gene system is its polymorphism. Each gene, MHC class I (A, B and C) and MHC class II (DP, DQ and DR) exists in different alleles. Current nomenclature for HLA alleles is designated by numbers, as described by Marsh et al.: Nomenclature for factors of the HLA system, 2010. Tissue Antigens 75:291 -455, herein specifically incorporated by reference. For HLA protein and nucleic acid sequences, see Robinson J, Barker DJ, Georgiou X, Cooper MA, Flicek P, Marsh SGE IPD-IMGT/HLA Database. Nucleic Acids Res. 2020 Jan 8;48(D1 ):D948-D955. doi: 10.1093/nar/gkz950 herein specifically incorporated by reference.

[0073] MHC context. The function of MHC molecules is to bind peptide fragments derived from pathogens or aberrant proteins derived from transformed cells, and display them on the cell surface for recognition by the appropriate T cells. Thus, T cell receptor recognition can be influenced by the MHC protein that is presenting the antigen. The term MHC context or restriction refers to the recognition by a TCR of a given peptide, when it is presented by a specific MHC protein.

[0074] Peptide ligands are peptide antigens against which an immune response involving T lymphocyte antigen specific response can be generated, including autoantigens. T cells recognize peptides processed and presented on the cell surface, where the protein from which the peptide is derived is not necessarily a cell-surface protein.

[0075] Sequencing platforms that can be used in the present disclosure include but are not limited to: pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, second- generation sequencing, nanopore sequencing, sequencing by ligation, or sequencing by hybridization. Preferred sequencing platforms are those commercially available from Illumina (RNA-Seq) and Helicos (Digital Gene Expression or “DGE”). “Next generation” sequencing methods include, but are not limited to those commercialized by: 1 ) 454/Roche Lifesciences including but not limited to the methods and apparatus described in Margulies et al., Nature (2005) 437:376-380 (2005); and US Patent Nos. 7,244,559; 7,335,762; 7,211 ,390; 7,244,567; 7,264,929; 7,323,305; 2) Helicos BioSciences Corporation (Cambridge, MA) as described in U.S. application Ser. No. 11/167046, and US Patent Nos. 7501245; 7491498; 7,276,720; and in U.S. Patent Application Publication Nos. US20090061439; US20080087826; US20060286566; US20060024711 ; US20060024678; US20080213770; and

US20080103058; 3) Applied Biosystems (e.g. SOLiD sequencing); 4) Dover Systems (e.g., Polonator G.007 sequencing); 5) Illumina as described US Patent Nos. 5,750,341 ; 6,306,597; and 5,969,119; and 6) Pacific Biosciences as described in US Patent Nos. 7,462,452; 7,476,504; 7,405,281 ; 7,170,050; 7,462,468; 7,476,503; 7,315,019; 7,302,146; 7,313,308; and US Application Publication Nos. US20090029385; US20090068655; US20090024331 ; and US20080206764. All references are herein incorporated by reference. Such methods and apparatuses are provided here by way of example and are not intended to be limiting.

[0076] "Antigen" or "immunogen" refers to any substance that stimulates an immune response. The term antigen includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extract, or fragments thereof, individually or in any combination thereof.

[0077] "Cellular immune response" or "cell mediated immune response" is one mediated by T-lymphocytes or other white blood cells or both, and includes the production of cytokines, chemokines, cytolytic proteins and similar molecules produced by activated T-cells, white blood cells, or both.

[0078] "Emulsifier" means a substance used to make an emulsion more stable.

[0079] "Emulsion" means a composition of two immiscible liquids in which small droplets of one liquid are suspended in a continuous phase of the other liquid.

[0080] "Immune response" in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and a cellular immune response to an antigen. Immune responses can usually be determined using standard immunoassays and neutralization assays, which are known in the art.

[0081] "Immunogenic" means evoking an immune or antigenic response. Thus an immunogenic composition would be any composition that induces an immune response.

[0082] "Pharmaceutically acceptable" refers to substances, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to- risk ratio, and effective for their intended use.

[0083] "Immunostimulatory composition", or “vaccine” refers to a composition that includes an antigen, as defined herein and may optionally further include an adjuvant, in which case it may be more conventionally referred to as a vaccine. Typically, the composition will comprise an immunogenic PTM Tau peptide such as SEQ ID NO:7, 8, 12, 107, 113, 114, or homologous peptide sequence thereof. The peptides may be synthesized in vitro or by recombinant methods and modified to comprise an acetylated lysine. Administration of the composition to a subject results in an increased responsive state of immune cells. The amount of a composition that is therapeutically effective may vary depending on the presence of antigen, the adjuvant, and the condition of the subject, and can be determined by one skilled in the art. The peptides may be conjugated to a nanoparticle, carrier protein, etc. to enhance activity, or packaged in a lipid formulation. These materials can be purchased commercially.

[0084] Adjuvants can be mineral salts, emulsions, microparticles, saponins, cytokines, microbial components/products, or liposomes, and include as examples aluminum hydroxide, aluminum phosphate, calcium phosphate, microparticles, nanoparticles, squalene, saponin, and substances acting on toll like receptors. For example, AS03 is an adjuvant system composed of a-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion. MF59 is another immunologic adjuvant that comprises a squalene emulsion. The dose of adjuvant administered may depend on whether an antigen is present, on the antigen with which it is used and the antigen dosage to be applied. It is also dependent on the intended species and the desired formulation. Usually, the quantity is within the range conventionally used for adjuvants. For example, adjuvants typically comprise from about 1 j g to about 1000 j g, inclusive, of a 1 -mL dose.

[0085] The adjuvant formulations can be homogenized or microfluidized. The formulations are subjected to a primary blending process, typically by passage one or more times through one or more homogenizers. Any commercially available homogenizer can be used for this purpose, e.g., Ross emulsifier (Hauppauge, N.Y.), Gaulin homogenizer (Everett, Mass.), or Microfluidics (Newton, Mass.). In one embodiment, the formulations are homogenized for three minutes at 10,000 rpm. Microfluidization can be achieved by use of a commercial mirofluidizer, such as model number 110Y available from Microfluidics, (Newton, Mass.); Gaulin Model 30CD (Gaulin, Inc., Everett, Mass.); and Rainnie Minilab Type 8.30H (Miro Atomizer Food and Dairy, Inc., Hudson, Wis.). These microfluidizers operate by forcing fluids through small apertures under high pressure, such that two fluid streams interact at high velocities in an interaction chamber to form compositions with droplets of a submicron size. In one embodiment, the formulations are microfluidized by being passed through a 200 micron limiting dimension chamber at 10,000+/-500 psi.

[0086] The routes of administration for the vaccine compositions include parenteral, oral, oronasal, intranasal, intratracheal, topical, etc. Any suitable device may be used to administer the compositions, including syringes, droppers, needleless injection devices, patches, and the like. The route and device selected for use will depend on the composition of the adjuvant, the antigen, and the subject, and such are well known to the skilled artisan.

[0087] The adjuvant compositions can further include one or more immunomodulatory agents such as, e.g., quaternary ammonium compounds (e.g., DDA), and interleukins, interferons, or other cytokines. These materials can be purchased commercially. The amount of an immunomodulator suitable for use in the adjuvant compositions depends upon the nature of the immunomodulator used and the subject. However, they are generally used in an amount of about 1 j g to about 5,000 j g per dose. For a specific example, adjuvant compositions containing DDA can be prepared by simply mixing an antigen solution with a freshly prepared solution of DDA. [0088] The adjuvant compositions can further include one or more polymers such as, for example, DEAE Dextran, polyethylene glycol, and polyacrylic acid and polymethacrylic acid (eg, CARBOPOL. RTM.). Such material can be purchased commercially. The amount of polymers suitable for use in the adjuvant compositions depends upon the nature of the polymers used. However, they are generally used in an amount of about 0.0001% volume to volume (v/v) to about 75% v/v. In other embodiments, they are used in an amount of about 0.001% v/v to about 50% v/v, of about 0.005% v/v to about 25% v/v, of about 0.01% v/v to about 10% v/v, of about 0.05% v/v to about 2% v/v, and of about 0.1 % v/v to about 0.75% v/v. In another embodiment, they are used in an amount of about 0.02 v/v to about 0.4% v/v. DEAE-dextran can have a molecular size in the range of 50,000 Da to 5,000,000 Da, or it can be in the range of 500,000 Da to 2,000,000 Da. Such material may be purchased commercially or prepared from dextran.

[0089] The adjuvant compositions can further include one or more Th2 stimulants such as, for example, Bay R1005™ and aluminum. The amount of Th2 stimulants suitable for use in the adjuvant compositions depends upon the nature of the Th2 stimulant used. However, they are generally used in an amount of about 0.01 mg to about 10 mg per dose. In other embodiments, they are used in an amount of about 0.05 mg to about 7.5 mg per dose, of about 0.1 mg to about 5 mg per dose, of about 0.5 mg to about 2.5 mg per dose, and of 1 mg to about 2 mg per dose. A specific example is Bay R1005™, a glycolipid with the chemical name "N-(2-deoxy-2-L-leucylamino-[3-D-glucopyranosyl)-N-octadecyl dodecanamide acetate." It is an amphiphilic molecule which forms micelles in aqueous solution.

[0090] Oil, when added as a component of an adjuvant, generally provides a long and slow release profile. In the present invention, the oil can be metabolizable or non-metabolizable. The oil can be in the form of an oil-in-water, a water-in-oil, or a water-in-oil-in-water emulsion.

[0091] Oils suitable for use in the present invention include alkanes, alkenes, alkynes, and their corresponding acids and alcohols, the ethers and esters thereof, and mixtures thereof. The individual compounds of the oil are light hydrocarbon compounds, i.e., such components have 6 to 30 carbon atoms. The oil can be synthetically prepared or purified from petroleum products. The moiety may have a straight or branched chain structure. It may be fully saturated or have one or more double or triple bonds. Some non-metabolizable oils for use in the present invention include mineral oil, paraffin oil, and cycloparaffins, for example.

[0092] The term oil is also intended to include "light mineral oil," i.e., oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil.

[0093] Metabolizable oils include metabolizable, non-toxic oils. The oil can be any vegetable oil, fish oil, animal oil or synthetically prepared oil which can be metabolized by the body of the subject to which the adjuvant will be administered and which is not toxic to the subject. Sources for vegetable oils include nuts, seeds and grains.

[0094] Other components of the compositions can include pharmaceutically acceptable excipients, such as carriers, solvents, and diluents, isotonic agents, buffering agents, stabilizers, preservatives, vaso-constrictive agents, antibacterial agents, antifungal agents, and the like. Typical carriers, solvents, and diluents include water, saline, dextrose, ethanol, glycerol, oil, and the like. Representative isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like. Useful stabilizers include gelatin, albumin, and the like.

[0095] Surfactants are used to assist in the stabilization of the emulsion selected to act as the carrier for the adjuvant and antigen. Surfactants suitable for use in the present inventions include natural biologically compatible surfactants and non-natural synthetic surfactants. Biologically compatible surfactants include phospholipid compounds or a mixture of phospholipids. Preferred phospholipids are phosphatidylcholines (lecithin), such as soy or egg lecithin. Lecithin can be obtained as a mixture of phosphatides and triglycerides by waterwashing crude vegetable oils, and separating and drying the resulting hydrated gums. A refined product can be obtained by fractionating the mixture for acetone insoluble phospholipids and glycolipids remaining after removal of the triglycerides and vegetable oil by acetone washing. Alternatively, lecithin can be obtained from various commercial sources. Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, cardiolipin, and phosphatidylethanolamine. The phospholipids may be isolated from natural sources or conventionally synthesized.

[0096] Non-natural, synthetic surfactants suitable for use in the present invention include sorbitan-based non-ionic surfactants, e.g. fatty-acid-substituted sorbitan surfactants, fatty acid esters of polyethoxylated sorbitol (TWEEN™), polyethylene glycol esters of fatty acids from sources such as castor oil; polyethoxylated fatty acid, polyethoxylated isooctylphenol/formaldehyde polymer, polyoxyethylene fatty alcohol ethers (BRU™); polyoxyethylene nonphenyl ethers (TRITON™), polyoxyethylene isooctylphenyl ethers (TRITON™ X).

[0097] As used herein, "a pharmaceutically-acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. The carrier(s) must be "acceptable" in the sense of being compatible with the other components of the compositions and not deleterious to the subject. Typically, the carriers will be sterile and pyrogen-free, and selected based on the mode of administration to be used. It is well known by those skilled in the art that the preferred formulations for the pharmaceutically acceptable carrier which comprise the compositions are those pharmaceutical carriers approved in the applicable regulations promulgated by the United States (US) Department of Agriculture or US Food and Drug Administration, or equivalent government agency in a non-US country. Therefore, the pharmaceutically accepted carrier for commercial production of the compositions is a carrier that is already approved or will be approved by the appropriate government agency in the US or foreign country.

[0098] The compositions optionally can include compatible pharmaceutically acceptable (i.e., sterile or non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others.

[0099] Antigen binding region (ABR). As used herein, the term ABR refers to a combination of variable heavy (VH and variable light (VL) polypeptides to associate to form a variable region domain, which may be an antibody sequence or a T cell receptor sequence (see, for example, Table 2. An ABR is the minimum antibody fragment that contains a complete antigen-recognition and binding site, or a minimum T cell receptor antigen binding fragment that contains a complete antigen-recognition and binding site. This region consists of heavy- and one light-chain variable domain in tight, non-covalent association, as a single polypeptide or as a dimer. In an antibody it is in this configuration that the three CDRS of each variable domain interact to define an antigen-binding site on the surface of the domain. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[00100] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a [3-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the [3-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991 )). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[00101] An antibody or ABR “which binds” an antigen of interest, is one that binds the antigen with sufficient affinity such that the antibody or binding molecule is useful as a diagnostic and/or therapeutic agent in targeting the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody or other binding molecule to a non-targeted antigen will usually be no more than 10% as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[00102] Antibodies, also referred to as immunoglobulins, conventionally comprise at least one heavy chain and one light, where the amino terminal domain of the heavy and light chains is variable in sequence, hence is commonly referred to as a variable region domain, or a variable heavy (VH) or variable light (VH) domain. The two domains conventionally associate to form a specific binding region, although as well be discussed here, a variety of non-natural configurations of antibodies are known and used in the art.

[00103] The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain only antibodies, three chain antibodies, single chain Fv, nanobodies, etc., and also include antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour, of Immunology 170:4854-4861 ). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[00104] The term antibody may reference a full-length heavy chain, a full length light chain, an intact immunoglobulin molecule; or an immunologically active portion of any of these polypeptides, i.e., a polypeptide that comprises an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., lgG1 , lgG2, lgG3, lgG4, Ig A1 and lgA2) or subclass of immunoglobulin molecule, including engineered subclasses with altered Fc portions that provide for reduced or enhanced effector cell activity. The immunoglobulins can be derived from any species. In one aspect, the immunoglobulin is of largely human origin.

[00105] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region may comprise amino acid residues from a “complementarity determining region” or “CDR”, and/or those residues from a “hypervariable loop”. “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.

[00106] Variable regions of interest include CDR sequences, which may be obtained from available antibodies with the desired specificity, or may be obtained from antibodies developed for this purpose. One of skill in the art will understand that a number of definitions of the CDRs are commonly in use, including the Kabat definition (see “Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010;47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of the structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989;342:877-883). Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001 ;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008;181 :6230-6235; Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132-143; and Padlanet al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139., each of which is herein specifically incorporated by reference.

[00107] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[00108] The antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81 :6851-6855).

[00109] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “classes.” There are five major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, IgA, and lgA2. The heavychain constant domains that correspond to the different classes of antibodies are called a, 5, E, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modifications or hingeless forms (Roux et al (1998) J. Immunol. 161 :4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005/0048572; US 2004/0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called K and A, based on the amino acid sequences of their constant domains.

[00110] Effector CAR-T cells include autologous or allogeneic immune cells having immune activity against a target cell expressing an antigen of interest, may be Treg CART with suppressive activity, or T helper cells. The effector cells can have cytolytic activity that does not require recognition through the T cell antigen receptor. In some embodiments, a T cell is engineered to express a CAR. In some embodiments, a T cell is engineered to express an introduced T cell receptor sequence. The term “T cells” refers to mammalian immune effector cells that may be characterized by expression of CD3 and/or T cell antigen receptor.

[00111] In other embodiments, the engineered T cell is allogeneic with respect to the individual that is treated, e.g. see clinical trials NCT03121625; NCT03016377; NCT02476734; NCT02746952; NCT02808442. See for review Graham et al. (2018) Cells. 7(10) E155. In some embodiments an allogeneic engineered T cell is fully HLA matched. However not all patients have a fully matched donor and a cellular product suitable for all patients independent of HLA type provides an alternative. A universal ‘off the shelf’ CAR T cell product provides advantages in uniformity of harvest and manufacture.

[00112] Allogeneic T cells can be genetically modified to reduce graft v host disease. For example, the TCRap receptor can be knocked out by different gene editing techniques. TCRap is a heterodimer and both alpha and beta chains need to be present for it to be expressed. A single gene codes for the alpha chain (TRAC), whereas there are 2 genes coding for the beta chain, therefore TRAC loci KO has been deleted for this purpose. A number of different approaches have been used to accomplish this deletion, e.g. CRISPR/Cas9; meganuclease; engineered l-Crel homing endonuclease, etc. See, for example, Eyquem et al. (2017) Nature 543:113-117, in which the TRAC coding sequence is replaced by the CAR coding sequence; and Georgiadis et al. (2018) Mol. Ther. 26:1215-1227, which linked CAR expression with TRAC disruption by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 without directly incorporating the CAR into the TRAC loci. An alternative strategy to prevent GVHD modifies CAR-T cells to express an inhibitor of TCRap signaling, for example using a truncated form of CD3^ as a TCR inhibitory molecule. [00113] Allogeneic T cells may be administered in combination with intensification of lymphodepletion to allow CAR-T cells to expand and clear malignant cells prior to host immune recovery, e.g. by administration of Alemtuzumab (monoclonal anti-CD52), purine analogs, etc. The allogeneic T cells may be modified for resistance to Alemtuzumab, and currently in clinical trials. Gene editing has also been used to prevent expression of HLA class I molecules on CAR-T cells, e.g. by deletion of [32-microglobulin, see NCT03166878.

[00114] In addition to modifying T cells, induced pluripotent stem (iPS) CAR-T cells can provide a source of allogeneic CAR-T cells. For example, transducing donor T cells with reprogramming factors can restore pluripotency and are then re-differentiated to T effector cells.

[00115] T cells for engineering as described above collected from a subject or a donor may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation. An appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.

[00116] Expression construct: A protein (e.g. CAR, antibody, Fc fusion, Tau peptide, etc.) coding sequence may be introduced on an expression vector into a cell to be engineered. The nucleic acid encoding an ARB sequence is inserted into a vector for expression and/or integration. Many such vectors are available. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, and the like.

[00117] For example, a CAR coding sequence may be introduced into the site of the endogenous T cell receptor, e.g. TRAC gene, e.g., using CRISPR technology (see, for example Eyquem et al. (2017) Nature 543:113-117; Ren et al. (2017) Protein & Cell 1 -10; Ren et al. (2017) Oncotarget 8(10):17002-17011). CRISPR/Cas9 system can be directly applied to human cells by transfection with a plasmid that encodes Cas9 and sgRNA. The viral delivery of CRISPR components has been extensively demonstrated using lentiviral and retroviral vectors. Gene editing with CRISPR encoded by non-integrating virus, such as adenovirus and adenovirus-associated virus (AAV), has also been reported. Recent discoveries of smaller Cas proteins have enabled and enhanced the combination of this technology with vectors that have gained increasing success for their safety profile and efficiency, such as AAV vectors. [00118] Screening agents for monitoring and detection of T cells involved in anti-Tau immune responses are provided, including MHC tetramers/multimers with DRB1 *04:01 , DRB1 *04:02, DRB1 *04:03, DRB1 *04:04, DRB1 *04:05, DRB1 *04:06, DRB1 *04:07, DRB1 *04:08,

DRB1 *04:09, DRB1 *04:10, DRB1 *04:11 , DRB1 *04:12, DRB1 *04:13, DRB4*01 :01 ,

DRB4*01 :03 in combination with a Tau peptide, e.g. any of SEQ ID NO:66-109 and 192-193. DRB1 *04:01 and DRB1 *04:04 tetramers presenting a PTM Tau peptide comprising acetylated K311 or K280 (or mimics such as Q, glutamine) are of particular interest. The tetramers are useful in determining the presence of reactive T cells to Tau aggregation motifs and for isolating and characterizing the associated T cell phenotype. In some embodiments a method is provided for screening T cells from an individual for the presence of a protective response, the method comprising binding a T cell population to an MHC tetramer presenting a PTM Tau peptide comprising acetylated K311 or K280, and determining the number and/or frequency of binding T cells in the population. The number and/or frequency may be compared to a control population. The individual may be treated accordingly, where increased numbers of protective T cells indicate less risk of developing neurodegenerative diseases such as AD, PD or ALS.

[00119] MHC Tetramers are complexes of four MHC molecules, associated with a specific peptide and bound to a fluorochrome. MHC Tetramers may be generated essentially as described by Altman et al. Purified recombinant MHC (heavy chain) and human [32- microglobulin (|32m) are refolded in molar excess of the appropriate 8- to 10-mer peptide for several days. The refolded monomer can be biotinylated with a single biotin by the BirA enzyme at the C-terminal end of the heavy chain. The biotinylated monomers are purified by streptavidin-agarose affinity column chromatography. Subsequently, purified monomers are linked by the addition of labeled streptavidin.

[00120] By “cognition” it is meant the mental processes that include attention and concentration, learning complex tasks and concepts, memory (acquiring, retaining, and retrieving new information in the short and/or long term), information processing (dealing with information gathered by the five senses), visuospatial function (visual perception, depth perception, using mental imagery, copying drawings, constructing objects or shapes), producing and understanding language, verbal fluency (word-finding), solving problems, making decisions, and executive functions (planning and prioritizing). Cognition is a faculty for the processing of information, applying knowledge, and changing preferences. By “cognitive plasticity” it is meant the ability to learn, e.g., the ability to learn complex tasks and concepts, analogous to the ability to learn of an organism that is undifferentiated such as a newborn or juvenile, e.g., a human from the time of birth to pre-pubertal age of about 10 years. [001 1] By “cognitive decline”, it is meant a progressive decrease in cognition, as evidenced by, for example, a decline in one or more of, e.g., attention and concentration, learning complex tasks and concepts, memory (acquiring, retaining, and retrieving new information in the short and/or long term), information processing (dealing with information gathered by the five senses), visuospatial function (visual perception, depth perception, using mental imagery, copying drawings, constructing objects or shapes), producing and understanding language, verbal fluency (word-finding), solving problems, making decisions, and executive functions (planning and prioritizing).

[00122] By “an impairment in cognitive ability”, “reduced cognitive function”, and “cognitive impairment”, it is meant a reduction in cognitive ability relative to a healthy individual, e.g. an age-matched healthy individual, or relative to the ability of the individual at an earlier point in time, e.g. 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 5 years, or 10 years or more previously.

Conditions for treatment

[00123] Neurodegenerative diseases involving Tau. This refers to neurodegenerative diseases characterized by the deposition of abnormal tau protein that form neurofibrillary or gliof ibrillary tangles (NFTs) or other Tau aggregates in the brain. A variety of neurodegenerative diseases involving Tau may be treated by practicing the methods, including AD, PD, ALS, Pick’s disease, Lewy Body Dementia, progressive supranuclear palsy, corticobasal degeneration, Frontotemporal dementias, chronic traumatic encephalopathy (GTE) or post head trauma treatment aiming at preventing tau aggregation in these patients, argyrophilic grain disease, globular glial Tauopathies, , Vacuolar Tauopathy, Lytico-bodig disease, postencephalitic parkinsonism, subacute sclerosing panencephalitis and primary age-related Tauopathy, which aging-related Tau astrogliopathy.

[00124] Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with memory loss, spatial disorientation, and gradual deterioration of intellectual capacity. Numerous pathological changes have been described in the postmortem brains of AD patients, including synaptic and neuronal loss, oxidative damage, activated inflammatory cells, amyloid plaques mainly composed of the B-amyloid peptide (AB), and neurofibrillary tangles (NFTs) comprised of hyperphosphorylated and/or acetylated aggregates of the microtubule- associated protein Tau, the latter two of which are considered the pathological hallmarks. For several reasons, research on the involvement of AB in AD has progressed more quickly than that on Tau. The description of the “amyloid cascade hypothesis” based on the discovery of genetic mutations that cause autosomal familial AD centered the focus of research on AB. Also, the biochemical studies of amyloid precursor protein (APP) and the presenilins have greatly enhanced the understanding of the molecular pathways leading to AB generation. These studies favored the systematic development of disease-modifying therapies based on AB pathway.

[001 5] The classically described function of Tau is as a neuronal microtubule-associated protein, mainly found in axons. Under physiological conditions, Tau exists as a highly soluble and natively unfolded protein that interacts with tubulin and promotes its assembly into microtubules, which helps to stabilize their structure. Recent evidence points to additional functions for Tau. For example, Tau phosphorylation enables neurons to escape from an acute apoptotic death through stabilizing B-catenin. Also, Tau exerts an essential role in the balance of microtubule-dependent axonal transport of organelles and biomolecules by modulating the anterograde transport by kinesin and the dynein-driven retrograde transport.

[00126] In the healthy brain, 2-3 residues on Tau are phosphorylated. In AD and other Tauopathies, however, the phosphorylation level of Tau is significantly higher, with approximately nine phosphates per molecule. Hyperphosphorylation of Tau may occur at different putative serine, threonine, and tyrosine residues through a disruption in the equilibrium of Tau kinases and Tau phosphatases activity. The consequences of this phenomenon are still under investigation, but produces the outcome of lowering Tau's affinity for the microtubules as well as increasing Tau's resistance to calcium-activated neutral proteases and its degradation by the ubiquitin-proteosome pathway. Ultimately, Tau hyperphosphorylation leads to fibrillization and aggregation into NFTs. The major Tau kinases include glycogen-synthase kinase-3B (GSK-3B), cyclin-dependent protein kinase 5 (CDK5), cAMP-dependent protein kinase (PKA), mitogen-activated protein kinases (MAPK), calcium- calmodulin-dependent kinase-ll (CaMK II), and microtubule affinity-regulating kinase (MARK). Among the phosphatases, protein phosphatase 2 (PP2A) has been most implicated in the dephosphorylation of abnormal Tau. Notably, changes in the expression and/or activation of Tau kinases and Tau phosphatases have been well documented in AD and related disorders. Studies in transgenic mouse models of AD suggest that it is likely that multiple, overlapping processes contribute to abnormal hyperphosphorylation of Tau, including AB, impaired brain glucose metabolism, and inflammation.

[00127] The traditional view of Tau pathogenesis has focused predominantly on hyperphosphorylated Tau, but mounting evidence now implicates aberrant Tau acetylation on lysine residues spanning the microtubule (MT) binding repeat region (MTBR) as another contributing factor. The sheer abundance of Tau acetylation within the MTBR (>25 lysine residues identified) and the ability of this modification to control Tau binding to MTs, Tau aggregation, and Tau oligomer formation suggest that aberrant Tau acetylation acts in a pathological manner to promote neurotoxicity and cognitive decline. Supporting this notion, recent studies indicate that acetylated Tau is also sufficient to promote synaptic degeneration and cognitive dysfunction. Analysis of diseased human postmortem brain tissues also shows robust accumulation of acetylated Tau within neurofibrillary tangles (NFTs) and other aggregated Tau lesions. For example, using site-specific Tau acetylation antibodies, acetylated Tau was found in AD brains and in a range of other neurodegenerative diseases involving Tau including Pick’s disease (PiD), corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP). In contrast to phosphorylated Tau, acetylated Tau at residue K280 (ac-K280) is undetectable in cognitively normal control brains, suggesting ac- K280 is a disease-specific marker for Tauopathies. This concept has important clinical implications since acetylated Tau could be directly linked to Tauopathy onset, Tau strain specificity, and/or disease progression. Tau acetylation may facilitate the formation of toxic Tau oligomers that lead to synaptic dysfunction. A K174Q acetylation-mimic mutant showed impaired Tau turnover and induced cognitive deficits. Similarly, K274/K281 Q acetylation mimic transgenic mice showed altered levels of critical synaptic factors (e.g., KIBRA), altered postsynaptic remodeling, and impaired long-term potentiation leading to memory deficits. In addition to ac_K280, a potential 3R-Tau specific strain (ac-K31 1 Tau) was identified that is prominent in AD and PiD, but not in CBD, PSP, and FTLD-Tau cases harboring 4R-Tau, even though the K31 1 residue is present in all six Tau isoforms capable of forming Tau inclusions. With K317 located nearby, the K31 1 acetylation of PHF6 is the most differentiating Tau PTM found in AD versus control brains. Further, K311 acetylation has been shown by multiple investigators to promote aggregation of PHF6 in vivo, in vitro and in silica, while K311 carbamylation is inhibitory. Crystalography studies have also shown that acetylated PHF6 dominates in the formation of long fibrils as in neurofibrillary tangles of AD. Acetylation at the K311 Tau residue may be mediated by SIRT1 and/or HDAC6, current therapeutic targets in AD. Similarly, recent evidence suggests that reducing acetylated Tau is neuroprotective in brain injury. K31 1 is not only acetylated, but also ubiquitinated, or succinylated, and the epitope trafficked to the NLRP3 inflammasome of microglial cells, where HLA class II presentation of Tau fragments by HLA-DRB1 *04 to T cells is also likely to occur.

[001 8] Soluble oligomeric species of amyloid-p (A|3) are thought to be other key mediators of cognitive dysfunction in Alzheimer’s disease (AD) (M. Sheng, et al. (2012) Cold Spring Harb Perspect Biol 4; J. J. Palop et al. (2010) Nat Neurosci 13, 812). Neuritic plaques, a hallmark of Alzheimer’s Disease, are accumulations of aggregated, or oligomerized, amyloid beta (A|3) peptides, including A|31 -40 (A|340) and A|31 -42 (A|342) that are derived from the processing of amyloid precursor protein (APP) by |3- and y-secretases. The vast majority of autosomal familial AD (FAD)-linked mutations are associated with increased levels of Ap 1 -42. T ransgenic mice expressing elevated levels of human A|3 experience memory loss and synaptic regression (M. Faizi et al., (2012) Brain Behav 2, 142; C. Perez-Cruz et al., (201 1 ) J Neurosci 31 , 3926; S. Knafo et al., (2009) Cereb Cortex 19, 586; M. Cisse et al., (201 1 ) Nature 469, 47). Ap production is thought to be activity-dependent (F. Kamenetz et al., (2003) Neuron 37, 925; J. Wu et al., (2011 ) Cell 147, 615), and even in wild type mice addition of soluble A|3 oligomers to hippocampal slices or cultures induces loss of long-term 2 potentiation (LTP), increases long-term depression (LTD) and decreases dendritic spine density (G. M. Shankar et al., (2007) J Neurosci 27, 2866; G. M. Shankar et al., (2008) Nat Med 14, 837; H. Hsieh et al., (2006) Neuron 52, 831 ). There are currently no effective therapies for arresting or reversing the impairment of cognitive function that characterizes AD.

[00129] Parkinson's disease (PD) is a major neurodegenerative disease that primarily affects motor systems but can also be accompanied by cognitive and behavioral problems. There is a widespread neuron degeneration in PD brains, affecting up to 70% of dopaminergic neurons in the substantia nigra (SN) by the time of death. The neuropathological hallmarks of PD include Lewy bodies (LBs) in the SN, brainstem, and rostral and forebrain regions and the selective deletion of dopaminergic neurons in the SN. Cell-death induced damage in SN may be the source of patient movement disorders. Although the causes of this cell death are generally unclear, researchers have observed an enrichment alpha-synuclein in neuronal Lewy bodies. Tau aggregates can also be observed in PD, notably in cases with LKKR2 mutations. Tau has also been associated with increased alpha synuclein deposits. Immunohistochemistry with anti-tau antibodies showed high level of NFTs in the substantia nigra from post-mortem human brain tissue. Researchers have also reported that tauopathies in PD and PD with dementia (PDD) were only observed in DA neurons of the nigrostriatal region, which contrasts with the wide-spread expression pattern of tau throughout the entire brain in AD.

[00130] In Parkinson disease, pigmented neurons of the substantia nigra, locus coeruleus, and other brain stem dopaminergic cell groups degenerate. Loss of substantia nigra neurons results in depletion of dopamine in the dorsal aspect of the putamen (part of the basal ganglia) and causes many of the motor manifestations of Parkinson disease.

[00131] A genetic predisposition is likely in at least in some cases of Parkinson disease. A genetic association with polymorphisms surrounding the tau gene is found in Parkinson disease and Alzheimer’s dementia. About 10% of PD patients have a family history of Parkinson disease. Several abnormal genes have been identified. Inheritance is autosomal dominant for some genes and autosomal recessive for others. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most prevalent mutation in sporadic cases of Parkinson disease in patients, and it is the most prevalent autosomal dominant mutation of the inherited forms of the disease. Recent data shows that tau is particularly important in PD patient with LRRK2 mutation, thus the therapy proposed here could be particularly effective.

[00132] Diagnosis of Parkinson disease is clinical. Parkinson disease is suspected in patients with characteristic unilateral resting tremor, decreased movement, or rigidity. During finger-to- nose coordination testing, the tremor disappears (or attenuates) in the limb being tested. During the neurologic examination, patients cannot perform rapidly alternating or rapid successive movements well. Sensation and strength are usually normal. Reflexes are normal but may be difficult to elicit because of marked tremor or rigidity. Slowed and decreased movement due to Parkinson disease must be differentiated from decreased movement and spasticity due to lesions of the corticospinal tracts. To help distinguish Parkinson disease from secondary or atypical parkinsonism, clinicians often test responsiveness to levodopa. A large, sustained response strongly supports Parkinson disease.

[00133] Levodopa is the most effective current treatment. However, when Parkinson disease is advanced, sometimes soon after diagnosis, response to levodopa can wear off, causing fluctuations in motor symptoms and dyskinesias. To reduce the time levodopa is taken and thus minimize these effects, clinicians can consider treating younger patients who have mild disability with MAO-B inhibitors (selegiline, rasagiline), Dopamine agonists (eg, pramipexole, ropinirole, rotigotine), Amantadine (which is also the best option when trying to decrease peakdose dyskinesias). However, if these drugs do not sufficiently control symptoms, clinicians should promptly initiate levodopa because it can usually greatly improve quality of life. Evidence now suggests that levodopa becomes ineffective because of disease progression rather than cumulative exposure to levodopa. [00134] Deep brain stimulation of the subthalamic nucleus or globus pallidus interna is often recommended for patients with levodopa-induced dyskinesias or significant motor fluctuations; this procedure can modulate overactivity in the basal ganglia and thus decrease parkinsonian symptoms in patients with Parkinson disease. For patients with tremor only, stimulation of the ventralis intermediate nucleus of the thalamus is sometimes recommended; however, because most patients also have other symptoms, stimulation of the subthalamic nucleus, which relieves tremor as well as other symptoms, is usually preferred. When the main problem is inadequate control of dyskinesias or when patients have an increased risk of cognitive decline, the globus pallidus interna is a good target.

[00135] .Amyotrophic lateral sclerosis is a group of rare neurological diseases that mainly involve the nerve cells (neurons) responsible for controlling voluntary muscle movement. It is characterized by steady, relentless, progressive degeneration of corticospinal tracts, anterior horn cells, bulbar motor nuclei, or a combination. Symptoms vary in severity and may include muscle weakness and atrophy, fasciculations, emotional lability, and respiratory muscle weakness. Diagnosis involves nerve conduction studies, electromyography, and exclusion of other disorders via MRI and laboratory tests. Current treatment is supportive. The majority of ALS cases (90 percent or more) are considered sporadic.

[00136] Most patients with ALS present with random, asymmetric symptoms, consisting of cramps, weakness, and muscle atrophy of the hands (most commonly) or feet. Weakness progresses to the forearms, shoulders, and lower limbs. Fasciculations, spasticity, hyperactive deep tendon reflexes, extensor plantar reflexes, clumsiness, stiffness of movement, weight loss, fatigue, and difficulty controlling facial expression and tongue movements soon follow. Other symptoms include hoarseness, dysphagia, and slurred speech; because swallowing is difficult, salivation appears to increase, and patients tend to choke on liquids. Late in the disorder, a pseudobulbar affect occurs, with inappropriate, involuntary, and uncontrollable excesses of laughter or crying. Sensory systems, consciousness, cognition, voluntary eye movements, sexual function, and urinary and anal sphincters are usually spared. Death is usually caused by failure of the respiratory muscles; 50% of patients die within 3 yr of onset, 20% live 5 yr, and 10% live 10 yr. Survival for > 30 yr is rare.

[00137] The drugs riluzole (Rilutek) and edaravone (Radicava) have been approved to treat certain forms of ALS. Riluzole is believed to reduce damage to motor neurons by decreasing levels of glutamate, which transports messages between nerve cells and motor neurons. Clinical trials in people with ALS showed that riluzole prolongs survival by a few months, particularly in the bulbar form of the disease, but does not reverse the damage already done to motor neurons. Edaravone has been shown to slow the decline in clinical assessment of daily functioning in persons with ALS. [00138] In ALS brain, aggregates of TDP-43 are typically found. There are also tau aggregates and Tau has been involved as a cofactor for the disease. In ALS, an HLA-DR4 protective association is also found as in AD and PD suggesting the same tau mediated protective effect is present.

[00139] Animal models for ALS include mutations in the SOD1 gene. Missense mutations in the SOD1 gene on chromosome 21 were the first identified causes of autosomal dominant FALS. SOD1 is a ubiquitous cytoplasmic and mitochondrial enzyme which functions in a dimeric state to catalyse the breakdown of harmful reactive oxygen species (ROS), thereby preventing oxidative stress. Sod1 ^_/ “ mice do not have any motor neuron loss, but they have a significant distal motor axonopathy, demonstrating the important role of SOD1 in normal neuronal function. The significant loss of motor neurons in transgenic mice expressing mutant SOD1 is likely to result from a toxic gain-of-function.

Anti-Tau Agents

[00140] In one aspect, this application is directed to anti-tau agents that inhibit the aggregation of acetylated tau protein, i.e. tau that is specifically acetylated at K280 or/and K311 . Such agents include antibodies, T cells, and the like as disclosed herein. The anti-tau agents may specifically bind to an acetylated tau protein to prevent the formation or disaggregate neurofibrillary tangles (NFTs) comprising acetylated tau proteins, or may generate an adaptive immune response against acetylated tau protein to prevent the formation or disaggregate neurofibrillary tangles (NFTs) comprising acetylated tau proteins.

[00141] Anti-tau agents may be generated using any suitable method. Suitable methods for the generation and screening of binding agents as anti-tau agents, include without limitation, immunization of dromedaries, immunization of rabbits, immunization of goats, immunization of sheep, immunization of horses, immunization of chickens, immunization of mice, immunization of rats, immunization of guinea pigs, immunization of camels, immunization of alpacas, immunization of sharks, yeast surface display, etc. When using immunization or yeast surface display, immunization and yeast display should be conducted using an epitope or peptide of acetylated tau, i.e. tau acetylated at K280 and K311. Yeast surface display has been successfully used to generate specific ISVs as shown in McMahon et al. (2018) Nature Structural Molecular Biology 25(3): 289-296 which is specifically incorporated herein by reference. Antibodies generated from experimental animals or yeast display may be further modified for use in humans by generating humanized antibodies or fragments thereof.

[00142] An anti-tau agent may be generated using any acetylated form of a tau protein. In some embodiments, an acetylated tau protein used as a protective immunogen comprises an immunogenic PTM Tau peptide such as SEQ ID NO:7, 8, 12, 107, 113, 114, or homologous peptide sequence thereof sequences. VQIVYacetvIKPVDLSK (SEQ ID NO: 7) or ²⁷⁵ VQIIN(acetylK)KLDLSNV ²⁸⁷ (SEQ ID NO: 8) or immunogenic fragments thereof are of particular interest.

[00143] Although traditionally Parkinson disease and its variant pathology Lewy Body Dementia is considered an alpha synuclein/Lewy Body pathology, and Amyotrophic lateral sclerosis is considered a disease involving TDP-43 aggregates, data indicates that Tau is also involved in these diseases as well as in many other neurodegenerative diseases as well as following head trauma. Cryo EM studies have found that Tau aggregates found following head trauma are almost identical to those found in ADand involve PHF6. Tau aggregates in CBD, FLTD and other Taupathies are different but also involve PHF6 or PHF6-like sequences.

Methods of Treatment

[00144] The present disclosure provides methods for treating neurodegenerative diseases involving Tau, such as AD, PD, ALS, corticobasal dementia, frontotemporal dementia, or brain damage following head trauma. The methods comprise administering to the subject an effective amount of an agent that is an anti-tau agent as a single agent or combined with an additional one or more agent(s). Methods of treatment may include determining the effectiveness of therapy by monitoring clinical indicia for stabilization or reduction of adverse disease symptoms.

[00145] In dome embodiments, e.g. in the administration of Tau peptide vaccines, individuals that may receive particular benefit of the treatments herein have at least one HLA-DRB1 *04 allele. The individual may have any subtype of HLA-DRB1 *04 such as DRB1 *04:01 , DRB1 *04:02, DRB1 *04:03, DRB1 *04:04, DRB1 *04:05, DRB1 *04:06, DRB1 *04:07, DRB1 *04:08, DRB1 *04:09, DRB1 *04:10, DRB1 *04:11 , DRB1 *04:12, or DRB1 *04:13. Individuals that would receive the most benefit from the treats disclosed herein would include HLA-DRB1 *04 subtype DRB1 *04:04, DRB1 *04:07, DRB1 *04:01 , DRB1 *04:02, and DRB1 *04:03. Administration of antibodies, CART cells, etc. do not require the HLA-DRB1 *04 phenotype.

[00146] Any method of HLA typing may be used to select an individual comprising an HLA DRB1 *04 subtype. Methods of HLA typing, include without limitation, SSP- (sequence-specific primer), SSO- (sequence-specific oligonucleotide), RFLP-PCR (restriction fragment length polymorphism polymerase chain reaction), sequence-based typing (SBT), etc. Methods of HLA typing are disclosed in various publications such as Tait et al (Nephrology (Carlton). 2009 Apr;14(2):247-54), Bontadini (Methods. 2012 Apr;56(4):471-6.), or Erlich et al (BMC Genomics. 2011 Jan 18;12:42.), all of which are expressly incorporated by reference.

[00147] In some embodiments, including without limitation administration of a tau peptide vaccine containing the K280 or K311 acetylated epitope or a mimic of, for example with a glutamine or asparagine substitution, administration may be systemic, e.g. parenteral. In other embodiments, e.g. administration of a tau peptide binding agent, administration may be localized to the brain.

[00148] In another embodiment, a drug is added that allows HLA subtypes other than DRB1 *04 to bind and recognize acetyl PHF6 or similar sequences, creating a similar protective response as the one found in subjects with DRB1 *04.

[00149] Numerous localized drug delivery strategies have been developed to circumvent the blood brain barrier. For example, the insertion of polymeric implants that release drugs slowly into the surrounding tissue has been reported to be successful in treating tissues locally. Treatment may be enhanced by alternative delivery methods that increase the penetration distance of the drug into tissue and eliminate the rapid decay in concentration with distance that is characteristic of diffusion mediated transport. Convection-enhanced drug delivery (CED) uses direct infusion of a drug-containing liquid into tissue so that transport is dominated by convection. By increasing the rate of infusion, the convection rate can be made large compared with the elimination rate in a region of tissue about the infusion point. Thus, CED has the potential of increasing the drug penetration distance and mitigating the decay in concentration with distance from the release point.

[00150] Nanoparticles as drug or gene carriers may be used, e.g. in combination with CED. Transport of particles through the extracellular space of tissues is hindered by the large size of nanoparticles (10-100 nm), which are much larger than small molecule drugs or therapeutic proteins that more easily penetrate the brain extracellular matrix (ECM). However, nanoparticles may be able to penetrate brain tissue provided that particles are less than 100 nm in diameter, are neutral or negatively charged, and are not subject to rapid elimination mechanisms.

[00151] In some embodiments the agent is delivered as continuous intraventricular CNS administration. In some embodiments, intraventricular administration is combined with systemic administration, for example utilizing an implantable device to deliver the agent. In some embodiments the implantable device is an osmotic pump. The device may be implanted intraventricularly, for example, with a conventional stereotaxic apparatus.

[00152] A continuous delivery device includes, for example, an implanted device that releases a metered amount of an agent continuously over a period of time. The device may be implanted so as to release the anti-agent into the cerebrospinal fluid (CSF). An example of such devices is an osmotic pump, which operates because of an osmotic pressure difference between a compartment within the pump, called the salt sleeve, and the tissue environment in which the pump is implanted. The high osmolality of the salt sleeve causes water to flux into the pump through a semipermeable membrane which forms the outer surface of the pump. As the water enters the salt sleeve, it compresses the flexible reservoir, displacing the test solution from the pump at a controlled, predetermined rate. The rate of delivery is controlled by the water permeability of the pump’s outer membrane. Thus, the delivery profile of the pump is independent of the drug formulation dispensed. Drugs of various molecular configurations, including ionized drugs and macromolecules, can be dispensed continuously in a variety of compatible vehicles at controlled rates.

[00153] In certain embodiments the anti-tau agent is combined with a therapeutic dose of a drug used to treat AD,PD or ALS such as carbidopa/levodopa, anticholinergic drugs (e.g. pramipexole, ropinirole), MAO-B inhibitors (selegiline, rasagiline), amantadine, Dopamine agonists (e.g., pramipexole, ropinirole, rotigotine), catechol O-methyltransferase (COMT) inhibitors (eg entacapone, opicapone, tolcapone), aducanumab, donepezil, rivastigmine, memantine, manufactured combination of memantine and donepezil, or galantamine. Other combined agents may be targeting TDP43, alpha-synuclein or beta amyloid. The active agents may be administered in separate formulations, or may be combined, e.g. in a unit dose. The formulation may be for oral administration, for local intraventricular or CED administration, etc.

[00154] In some embodiments the combined therapies are administered concurrently, where the administered dose of any one of the compounds may be a conventional dose, or less than a conventional dose. In some embodiments the two therapies are phased, for example where one compound is initially provided as a single agent, e.g. as maintenance, and where the second compound is administered during a relapse, for example at or following the initiation of a relapse, at the peak of relapse, etc.

[00155] In various aspects and embodiments of the methods and compositions described herein, administering the therapeutic compositions can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intravenously, orally, via implant, transmucosally, transdermally, intramuscularly, intrathecally, and subcutaneously. The delivery systems employ a number of routinely used pharmaceutical carriers.

[00156] In methods of use, an effective dose of an anti-tau agent of the invention is administered alone, or combined with additional active agents for the treatment of a condition as listed above. The effective dose may be from about 1 ng/kg weight, 10 ng/kg weight, 100 ng/kg weight, 1 j g/kg weight, 10 j g/kg weight, 25 j g/kg weight, 50 j g/kg weight, 100 j g/kg weight, 250 j g/kg weight, 500 j g/kg weight, 750 j g/kg weight, 1 mg/kg weight, 5 mg/kg weight, 10 mg/kg weight, 25 mg/kg weight, 50 mg/kg weight, 75 mg/kg weight, 100 mg/kg weight, 250 mg/kg weight, 500 mg/kg weight, 750 mg/kg weight, for example up to about 500 mg/kg weight, and the like. The dosage may be administered multiple times as needed, e.g. every 4 hours, every 6 hours, every 8 hours, every 12 hours, every 18 hours, daily, every 2 days, every 3 days, weekly, and the like. The dosage may be administered orally. [00157] The compositions can be administered in a single dose, or in multiple doses, usually multiple doses over a period of time, e.g. daily, every-other day, weekly, semi-weekly, monthly etc. for a period of time sufficient to reduce severity of the neurodegenerative diseases involving Tau, which can comprise 1 , 2, 3, 4, 6, 10, or more doses.

[00158] Determining a therapeutically or prophylactically effective amount of an agent according to the present methods can be done based on animal data using routine computational methods. The effective dose will depend at least in part on the route of administration.

[00159] All compounds of the invention are purified and/or isolated. Specifically, as used herein, an "isolated" or "purified" polypeptide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. Purified also defines a degree of sterility

Pharmaceutical Compositions

[00160] The above-discussed compounds can be formulated using any convenient excipients, reagents and methods. Compositions are provided in formulation with a pharmaceutically acceptable excipient(s). A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7 ^th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3 ^rd ed. Amer. Pharmaceutical Assoc.

[00161] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[00162] In some embodiments, the subject compound is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strengths from 5mM to 100mM. In some embodiments, the aqueous buffer includes reagents that provide for an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars e.g., mannitol, dextrose, sucrose, and the like. In some embodiments, the aqueous buffer further includes a non-ionic surfactant such as polysorbate 20 or 80. Optionally the formulations may further include a preservative. Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at about 4 ^e C. Formulations may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures. In some embodiments, the subject compound is formulated for sustained release.

[00163] In some embodiments, the anti-tau agent is formulated with a second agent in a pharmaceutically acceptable excipient(s).

[00164] The subject formulations can be administered orally, subcutaneously, intramuscularly, parenterally, or other route, including, but not limited to, for example, oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intravesical or injection into an affected organ.

[00165] Each of the active agents can be provided in a unit dose of from about 0.1 j g, 0.5 j g, 1 j g, 5 j g, 10 j g, 50 j g, 100 j g, 500 j g, 1 mg, 5 mg, 10 mg, 50, mg, 100 mg, 250 mg, 500 mg, 750 mg or more.

[00166] The anti-tau agent may be administered in a unit dosage form and may be prepared by any methods well known in the art. Such methods include combining the subject compound with a pharmaceutically acceptable carrier or diluent which constitutes one or more accessory ingredients. A pharmaceutically acceptable carrier is selected on the basis of the chosen route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. This carrier can be a solid or liquid and the type is generally chosen based on the type of administration being used.

[00167] Examples of suitable solid carriers include lactose, sucrose, gelatin, agar and bulk powders. Examples of suitable liquid carriers include water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions, and solution and or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Preferred carriers are edible oils, for example, corn or canola oils. Polyethylene glycols, e.g. PEG, are also good carriers.

[00168] Any drug delivery device or system that provides for the dosing regimen of the instant disclosure can be used. A wide variety of delivery devices and systems are known to those skilled in the art.

EXPERIMENTAL

[00169] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

Example 1

Protective effects of HLA-DRB1 *04 subtypes in Parkinson’s and Alzheimer’s diseases implicate Tau PHF6 in the pathophysiology of both diseases

[00170] To better understand the involvement of HLA in neurodegenerative diseases, GWAS data available in PD and AD was gathered, refining the signal to the HLA subtype level though HIBAG imputation and fine mapping studies. Because HLA signals are best dissected across ancestry groups, as first shown in narcolepsy with HLA-DQ, we included Europeans, Asians, Latinos and African Americans (FIG. 12 for samples involved). Here we show that subtypes of Human Leukocyte Antigen (HLA) DRB1 *04 protect against both PD and AD potentially by presenting acetylated epitopes of PHF6 sequences of the microtubule-associated protein Tau to CD4 ⁺ T cells. CD4 T cells recognizing these epitopes were also found in subjects with DRB1 *04, who are protected against these diseases. As these epitopes are known to seed Tau aggregation, we propose that improved immune clearance of Tau ensues, reducing pathology. Targeting this epitope through vaccination or immunotherapy may have preventive or therapeutic potential.

[00171] Colocalization of HLA SNP signals in AD and PD across ethnic groups. Two recent large studies in PD have shown that the HLA signal in PD is best summarized by a primary protective effect of DRB1 amino acids 13H and 33H shared by all DRB1 *04 subtypes. To extend on this, samples were meta-analyzed from North and South America, Europe, East Asia, expanding the sample size to a total of 61 ,203 PD and PD-proxy cases and 1 ,476,115 controls (FIG. 12). Asian samples were added because of the differential DRB1 *04 subtype distribution in Asians versus Europeans (key haplotypes distribution by ancestry, FIG. 4). The regional HLA association plot confirmed a primary effect driven by rs504594 (odds ratio (OR) = 0.84; 95% confidence interval [Cl] [0.80; 0.88]; p=1 .83x1 O' ¹³ ) in PD (locuszoom, FIG. 5), with high association with DRB1 *04-specific amino acid 13H/33H across all ancestry groups (lead variant linkage disequilibrium with various DRB1 *04 subtypes and key amino acids across ancestry groups, FIG. 6) and disappearance of the signal after rs504594 conditioning. This confirmed the initial findings in a larger and more diverse multi-ancestry sample. In contrast to PD, the HLA signal present in AD has not been finely characterized. To further examine this, the region was analyzed across diverse ancestry groups in 121 ,371 AD and AD-proxy cases and 410,989 controls from North America, Europe, Africa, and East Asia (FIG. 12 for description of subsamples). As shown in FIG. 5, the signal peaked at rs35472547 (OR = 0.91 ; 95% Cl [0.89; 0.93]; p= 9.7x1 O' ²³ ). The AD and PD signals colocalize in Europeans and across ancestry groups, with rs601945 as the best candidate representative of both signals (FIG. 1 , posterior probability of colocalization, PP4 = 99.5%). rs601945 correlates highly with the presence of DRB1 *04.

[00172] Fine mapping studies implicates protective effect of specific HLA-DRBTOA subtypes in AD and PD HLA imputation and HLA-DR-DQ analysis were next conducted in both diseases separately and jointly, as no heterogeneity was found (FIG. 10). As reported in PD, DRB1 13H or 33H (two DRB1 *04 amino acids in complete linkage disequilibrium) gave the highest association signal in both diseases. DQB1 *03:02, a subtype highly linked with DRB1 *04 (FIG. 4), was also associated, but less strongly (FIG. 10), suggesting a primary effect of DRB1 . Further, various DRB1 *04 subtypes had hierarchical effects on susceptibility. Specifically, DRB1 *04:04 and DRB1 *04:07, conferred the strongest protection, followed by weaker effects of DRB1 *04:01 and DRB1 *04:03 and no effect for DRB1 *04:05 and DQB1 *04:06 (FIG. 10). The absence of association was notable for DRB1 *04:05 which is a frequent HLA-DRB1*04 subtype in Asians. Although DRB1 *04:05 shares DRB1 *04 13H or 33H, it is distinct from common European DRB1 *04:01 , DRB1 *04:02, DRB1 *04:04 and Latino DRB1 *04:07 subtypes because of a D to S substitution at position 57. An additional weaker predisposing effect of DRB1 *01 :01 -DQA1 *01 :01 -DQB1 *05:01 was also observed in AD and was nominally significant in PD with the same direction of effect (FIG. 10). No other genome wide HLA significant effect was observed, notably with DRB1 *07:01 or DRB1 *09:01 , subtypes which are linked with accessory gene DRB4*01 also present on DRB1 *04 haplotypes. Of note, DRB1 is more expressed than other DRBs, DQ and DP, and is generally the immunodominant response of CD4 cells. Further, accessory DRB3/4/5 genes also commonly share a binding repertoire with primary DRB1 genes (as shown in FIG. 8)) and are typically minor components of HLA Class II immune responses. [00173] Tau-pathology is reduced in AD individuals with DRB1*04. Neuropathological information in 8,252 postmortem samples of European ancestry available through the Religious Orders Study and Memory and Aging Project and the National Institute on Aging - Alzheimer’s Disease Center cohorts 1 to 7 was first used, looking at the effect of rs601945, on tau Braak staging and neuritic plaques density in US-based pathological samples. As shown in FIG. 11 , a strong association of rs601945, and by extension DRB1 *04 H13 and DRB1 *04 alleles (FIG. 13) with neurofibrillary tangles but not amyloid plaques was observed, suggesting the involvement of Tau. The analysis of cerebrospinal fluid (CSF) A|342 and Tau levels int 6,139 subjects of European ancestry independently confirmed this observation. In CSF, rs601945 and DRB1 *04:04 were associated with a significant decrease of phosphorylated- and total- Tau (FIG. 11), but only weakly associated with increased A|342. Interestingly, DRB1 *04 H13 was also associated with an older age of onset in AD (FIG. 13).

[00174] The same locus is associated with decreased risk in other neurodegenerative diseases. The HLA locus association with AD and PD was found to colocalize with the protective HLA signal in amyotrophic lateral sclerosis (ALS), with PP4 =94.6%, and PP4=60.6%, respectively (FIG. 1 ). The lower level of colocalization in PD is due to the absence of the main lead variants in the latest ALS GWAS summary statistics (FIG. 5), and it was hypothesized that improved quality control and imputation on the TOPMed reference panel would strengthen this weak PP4 colocalization.

[00175] In addition, studying a subset of autopsied demented subjects with either Lewy Body or AD pathology or both pathologies, rs601945 shows a nominally significant protective association in the AD only pathology group and concordant protective effects in the Lewy Body only pathology group and in the dual pathology group (though smaller sample sizes here resulted in non-significant results)(FIG. 11). This protective association was also observed in the largest GWAS to date of Lewy body dementia, that included a mix of clinically diagnosed and pathologically confirmed cases with a suggestive but non-significant p-value (FIG. 11). Taken together, these results indicate that the HLA locus may be involved in a common immune response across these neurodegenerative diseases.

[00176] DRB1 *04 preferentially binds acetylated forms of the PHF6 sequences known to be aggregation seeds. Based on these results, it was hypothesized that a DRB1 *04- restricted adaptive immune response directed against Tau may be protective in AD and other neurodegenerative diseases. Tau, like other proteins involved in neurodegeneration, is highly post-translationally-modified (PTM) through phosphorylation and acetylation, phenomena that likely predispose to Tau aggregation. In autoimmune diseases, PTMs (such as citrullination of fibrinogen for rheumatoid arthritis) are frequently found in culprit autoantigens. Further, PTMs contribute to reduced self-tolerance as these are not presented in the thymus. This likely explains the strong polyclonal T cell response observed in controls and AD/PD patients against Tau, p-amyloid and a-synuclein. A similarly broad B cell response against Tau and a- synuclein is also reported in controls and patients. As mentioned above, prior work has outlined strong polyclonal CD4 ⁺ T cell responses against a-synuclein, p-amyloid and tau peptides when presented by various HLA subtypes in both cases and controls, thus it is unclear if these responses are effective in limiting disease or are a simple bystander effect. One recent publication also showed the presence of CD4 ⁺ and CD8 ⁺ T cells in the CSF of PD and AD patients, suggesting CD8 ⁺ T cell-mediated clearing of amyloid plaques.

[00177] Considering that CD4 ⁺ T cell and B cell responses against Tau are common and robust, it was thus hypothesized that a specific DRB1 *04 restricted response, unlike other responses, may target a particular Tau epitope potentially important for aggregation. To test this hypothesis, the most frequent PTM and non-PTM modified peptides for HLA-DRB1*04 subtype-specific binding, using the highly protective DRB1 *04:04 subtype, the moderately protective DRB1 *04:01 subtype, and the neutral DRB1 *04:05 subtype were screened (FIG. 2). Among over a thousand peptides tested, only a few peptides strongly bound DRB1 *04, with acetylated PHF6 (R3/wt, SEQ ID NO:8; ³⁰⁶ VQIVY(acetylK)PVDLSKV ³¹⁸ ) standing as the ideal candidate (FIG. 2). The PHF6 sequence is located within the Microtubule Binding Area of Tau known to be important for aggregation. PHF6 creates a core nucleation site that can, by themselves, induce formation of fibrils displaying the paired helical filament (PHF) morphology characteristic of neurofibrillary tangles. Most interestingly, these sequences only bind DRB1 *04 when K280 or K31 1 are acetylated. Further, these sequences have significantly less affinity for DRB1 *04:05 versus other subtypes (DRB1 *04:04=DRB1 *04:01 >DRB1 *04:03> DRB1 *04:06>DRB1 *04:05), the same hierarchy observed in the case/control association results, with cores predicted to strongly bind DRB1 *04:04 (FIG. 2, FIG. 8). This is most likely because serine in molecular pocket P9 is known to reduce DRB1 *04:05 specificity. Additional PTMs in the area, such as acetylated K317 do not alter binding, although the presence of phosphoserine at S305, another frequent PTM, slightly reduces PHF6 binding (FIG. 2).

[00178] The fact that DRB1 *04 only binds acetylated forms of PHF6 sequences strongly favor involvement of these epitopes in the protective effect of DRB1 *04 in AD. With K317 located nearby, the K31 1 PTMs (acetylation, ubiquitination or both) of PHF6 are the most differentiating Tau PTMs found in AD versus control brains. Further, K31 1 acetylation has been shown by multiple investigators to promote aggregation of PHF6 in vivo, in vitro and in silico, while K31 1 carbamylation inhibits aggregation. Finally, DRB1 *04-associated subtypes are the only frequent HLA DR and DQ subtypes with predicted higher affinity for the acetylated epitope (FIG. 7), likely explaining why DRB1*04 and no other subtypes mediate this effect.

[00179] Acetylation at these Tau residues may be mediated by SIRT1 and/or HDAC6, current therapeutic targets in AD. Similarly, recent evidence suggests that reducing acetylated Tau is neuroprotective in brain injury. Various combinations of PHF6 may explain variability in Tau aggregate morphology across 3- or 4-repeat containing tauopathies, with acetylated PHF6 dominating formation of long fibrils as in neurofibrillary tangles of AD. K311 is not only acetylated, but also ubiquitinated, or succinylated, the epitope trafficked to the NLRP3 inflammasome of microglial cells, where HLA class II presentation of Tau fragments by DRB1 *04 to T cells is likely to occur. Involvement of microglial cells is also suggested by various GWAS association signals observed in AD and PD. Although the ubiquitinated K311 epitope is unlikely to bind DRB1 *04 due to steric hinderance at P4, ubiquitination at K317 at P10 could further modulate DRB1 *04 binding with the effect of K311 succinylation unknown. Additional experiments exploring DRB1 *04 subtype binding of PTM modified segments of PHF6 in various combinations will be needed to further this line of investigation.

[00180] Interestingly, besides PHF6, a homologous segment, PHF6* (R2/wt; SEQ ID NO:9 2 ⁷³ GKVQIINKKLDL ²⁸⁴ ) has similar pro-aggregation properties in vitro , with acetylation of K280 in PHF6* also promoting aggregation in vitro. Involvement of PHF6*, an earlier candidate for aggregation seeding, is however less clear in vivo in comparison to involvement of acetyl K311 -PHF6, as acetylation of K280 is a rarer PTM. Further, although predicted to also bind HLA-DRB1 *04 with a similar repertoire (FIG. 21 , with acetylated K280 in position 4), acetylated PHF6* sequences did not bind in vitro (FIG. 22), illustrating the limitation of in silica predictions. Modifications of PHF6* may however produce the same effect as PHF6 and as such are considering embodiments.

[00181] A less probable candidate is in the C-terminal end of Tau, the SEQ ID NO:11 4 ⁴⁸ TLADEVSAS ⁴⁵⁷ sequence. This sequence also binds DRB1 *04:04>DRB1 *04:01 >DRB1 *04:05 as expected from the association studies. This region may also be relevant to AD pathogenesis as C-terminal cleavage of Tau is an early event leading to aggregation. This C-terminal epitope is, however, also predicted to bind other frequent subtypes such as DRB1 *03:01 and DRB1*01 :01 with similar affinity and core sequence and is thus a less likely candidate. Further, no T cells recognizing this epitope were detected in DRB1 *04 individuals (FIG. 20).

[00182] Overall, these results indicate that a DRB1 *04-subtype specific adaptive immune response is protective against both AD and PD, with recent work suggesting the same is true for ALS. Although it is impossible to exclude involvement of other proteins in this effect, a CD4 ⁺ T cell reactivity toward PHF6 fragments containing the acetylated K311 and K280 epitopes of Tau is a strong candidate for mediating this effect based on current knowledge. It was hypothesized that this T cell reactivity could facilitate early clearing of toxic tau aggregate seeds, for example by recruiting B cells and an antibody response targeting the same epitope (FIG. 3). Tau’s biology as a regulator of microtubule assembly in neurons is uniquely positioned to potentiate neurotoxicity across multiple diseases involving different aggregates, as reported here.

[00183] The results also show that targeting Tau epitopes containing acetyl-311 K and maybe acetyl-280K through chimeric antigen receptor T cells or antibodies could have therapeutic value. Further, vaccination with acetylated PHF6-like epitopes could reduce disease progression in DRB1 *04 individuals (-30% of the European population). It is noteworthy that antibodies, although not targeting acetyl-K311 per se, but regions adjacent within PHF6, have been shown to reduce CSF tau and Tau pathology in animal models and are being tested as a means of preventing autosomal dominant forms of AD. Autoantibodies against Tau are also present in healthy and demented individuals, but epitope specificities have yet to be determined. HLA studies of additional tauopathies with different aggregates may also help clarify how Tau modifications within the PHF6 or other aggregation-prone domains modulate other diseases. Tau may be an important modulator of neurodegeneration across multiple diseases.

METHODS

[00184] Samples Participants or their caregivers provided written informed consent in the original studies. The current study protocol was granted an exemption by the Stanford University institutional review board because the analyses were carried out on deidentified, off-the-shelf data; therefore, additional informed consent was not required.

[00185] The AD samples included in the analysis are part of the following datasets with phenotype, genotype ascertainment, and quality control described elsewhere: the European Alzheimer’s Disease BioBank (EADB), The Genome Research @ Fundacid ACE project (GR@ACE), Genetic and Environmental Risk in AD/Defining Genetic, Polygenic and Environmental Risk for Alzheimer’s Disease Consortium (GERAD/PERADES), the European Alzheimer’s Disease Initiative (EADI), the Norwegian DemGene (DemGene), the Bonn study (Bonn), the Copenhagen City Heart Study (CCHS), the Alzheimer's Disease Genetics Consortium (ADGC), the Alzheimer Disease Sequencing Project (ADSP), the UK Biobank, the Gwangju Alzheimer’s and Related Dementias (GARD) study, and the National Center for Geriatrics and Gerontology (NCGG) from Niigata University.

[00186] The PD samples included in the analysis are part of the following datasets for which the phenotyping, genotyping and quality control has been described elsewhere: International Parkinson’s Disease Genomics Consortium (IPDGC) NeuroX dataset, McGill University (McGill), National Institute of Neurological Disorders and Stroke (NINDS) Genome-Wide genotyping in Parkinson’s Disease, NeuroGenetics Research Consortium (NGRC), Oslo Parkinson’s Disease Study (Oslo), Parkinson’s Progression Markers Initiative (PPMI), Autopsy-Confirmed Parkinson Disease GWAS Consortium (APDGC), the UK Biobank, East Asians samples from Japan, China, Singapore, Taiwan, and Hong-Kong (EastAsians-PD), 23andMe, and the Latin American Research Consortium on the Genetics of Parkinson’s Disease (LARGE-PD).

[00187] The samples assessed for AD and Lewy-bodv neuropathology included genetic data from the Rush Religious Orders Study and Memory and Aging Project (ROSMAP) and from the Alzheimer’s Disease Center (ADC) cohorts 1 to 7 parts of the ADGC, and neuropathological assessment followed procedures described respectively in and in the National Alzheimer’s Coordinating Center (NACC) postmortem evaluation protocol.

[00188] Genome-wide association at the HLA locus and colocalization between AD and PD. Given the known signal at HLA in AD GWAS and PD GWAS. Refining the signal at the HLA locus using a multi-ancestry meta-analysis design was done. A region, ±1 MB around HLA-DRB1, on chromosome 6 from base pair positions (hg38) 31578952 to 33589718 was considered. For the PD local-GWAS at the HLA locus, the summary statistics from the largest available GWAS to date in European ancestry was meta-analyzed (distributed without 23andMe), with the Latino-Amerindian GWAS from (Loesch, D. P. et al.) and the Asian GWAS from (Foo, J. N. et al.). For the AD local-GWAS at the HLA locus, the summary statistics the largest available GWAS to date in European ancestry was meta-analyzed (which did not include their Stage 2), with the Korean/Japanese GWAS from (Kang, S. et al.), the Japanese GWAS from (Shigemizu, D. et al.), and with in-house local-GWAS at the HLA locus on ADSP and ADGC data carried out by ancestry in European, Latino-Amerindian, African individuals, analyzed with a linear-mixed model as implemented in GENESIS (see Statistical analysis section) adjusted for 6 PCs and sex. All meta-analyses were implemented with a fixed-effect inverse variance weighted design implemented in METAL. Colocalization between the AD and PD HLA signals in these multi-ancestry meta-analyses was assessed using the Bayesian model implemented in coloc using default priors. The posterior probability of colocalization (PP4) between AD and PD associations on FIG. 1 was reported. Given the high linkage disequilibrium (r ² > 0.70) of the lead SNP at the HLA locus in the latest amyotrophic lateral sclerosis (ALS) GWAS with the lead SNPs in the local-GWAS at HLA in AD and PD, the colocalization of these two diseases with ALS were also tested using the same method as described above. [00189] Imputation and statistical analysis of HLA alleles, haplotypes, and amino acids. Two-field resolution alleles of HLA-A, HLA-B, HLA-C class I genes, and HLA-DPB1, HLA- DQA1, HLA-DQB1, and HLA-DRB1 class II genes were imputed using R package HI BAG v1.22 for the following dataset: EADB, GR@ACE, GERAD, EADI, DemGene, Bonn, OCHS, UK Biobank, IPDGC, NINDS, NGRC, McGill, Oslo, PPMI, APDGC, LARGE-PD, ADSP, ADGC, GARD, NCGG. When available, training sets specific to ancestry (European, East Asian, Latino, African) and genotyping array were used, either available through HIBAG or trained in-house as previously described.

[00190] In the allele-level analyses, alleles with an imputation posterior probability lower than 0.5 were considered as undetermined as recommended by HIBAG developers. Each allele was considered as a variant and analyzed under a dominant model; supplementary methods provide details on the analysis per cohort.

[00191] In the haplotype-level analyses, only individuals with non-missing allele genotypes were included in the haplotype level analysis. Three-locus HLA class I or class II haplotypes were determined using the haplo.em function from the R haplo. stats package. Only haplotypes with posterior probability >0.5 and a carrier frequency of >1% were included in the analysis. Each haplotype was considered as a variant and analyzed under a dominant model; supplementary methods provide details on the analysis per cohort.

[00192] In the amino-acid-level analyses, HIBAG was used to convert P-coded alleles to amino acid sequences for exon 2, 3 of HLA class I genes, and exon 2 of class II genes. Each amino acid was considered as a variant and analyzed under a dominant model; supplementary methods provide details on the analysis per cohort.

[00193] For the East Asians-PD and 23andMe cohorts, the HLA alleles, haplotypes, amino acids statistics were derived from GWAS summary statistics data using the DISH software as described in.

[00194] The allele-, haplotype-, amino-acid- level analyses were respectively meta-analyzed separately between the two neurodegenerative diseases, and across diseases, using a fixed- effect inverse variance weighted design implemented in METAL.

[00195] Tau peptide binding. Competition binding was previously described. In brief, Tau and alpha-synuclein peptides at different concentrations were incubated with DRB1 *04:01 , DRB1 *04:03, DRB1 *04:04, DRB1 *04:05, or DRB1 *04:06 (from the Emory University NIH core tetramer facility) for 3 days at 37°C together with biotinylated GAD or EBV (Bio-GAD, EBV). The reaction was quenched by adding neutralization buffer and then transferred into anti-DR antibody precoated a 96-well plate. DELFI A® time-resolved fluorescence (TRF) intensity was detected using a Tecan SPARK after adding Europium (Eu)-labelled streptavidin. Non-specific binding was removed through extensive wash. Each peptide was duplicated. Competitor Tau peptide with Eu TRF intensity that was lower than 25% and 25-50% of Bio-GAD or EBV epitope alone was considered strong binder and weak binder, respectively.

QUALITY CONTROL AND ANALYSIS PER DATASET

[00196] Alzheimer’s Disease - ADSP & ADGC datasets. Participants and sources of data. Phenotypic information and genotypes were obtained from publicly released genome-wide association study datasets assembled by the Alzheimer's Disease Genetics Consortium (ADGC) and derived from whole-genome sequencing (WGS) data generated by the Alzheimer Disease Sequencing Project (ADSP), with phenotype and genotype ascertainment described elsewhere. The cohorts' queried accession numbers, as well as the sequencing technology or single nucleotide polymorphism (SNP) genotyping platforms are described in FIG. 14 and 15. The microarray datasets are largely part of the ADGC and as such they will be referred thereafter as the ADGC.

[00197] Quality control procedures. Prior to ancestry, principal components and relatedness determination, and HLA-alleles imputation, in each cohort-platform, variants were excluded based on genotyping rate (< 95%), MAF < 1%, and Hardy-Weinberg equilibrium in controls (p < 10' ⁶ ) using PLINK v1 .9. gnomAD database-derived information was used to filter out SNPs that met one of the following exclusion criteria: (i) located in a low complexity region, (ii) located within common structural variants (MAF > 1%), (iii) multiallelic SNPs with MAF > 1% for at least two alternate alleles, (iv) located within a common insertion/deletion, (v) having any flag different than PASS in gnomADv.3, (vi) having potential probe polymorphisms. The latter are defined as SNPs for which the probe may have variable affinity due to the presence of other SNP(s) within 20 bp and with MAF > 1%. Individuals with more than 5% genotype missingness were excluded. Duplicate individuals were identified with KING and their clinical, diagnostic and pathological data (including age-at-onset of cognitive symptoms, age-at-examination for clinical diagnosis, age-at-last exam, age-at-death), as well as sex, race, and APOE genotype were cross-referenced across cohorts. Duplicate entries with irreconcilable phenotype or discordant sex were flagged for exclusion.

[00198] Ancestry determination. For each cohort, we first determined the ancestry of each individual with SNPWeights v2 using reference populations from the 1000 Genomes Consortium. By applying an ancestry percentage cut-off > 75%, the samples were stratified into five super populations: South-Asians, East-Asians, Amerindians, Africans, and Europeans, and an Admixed group composed of individuals not passing the 75% cut-off in any single ancestry (FIG. 15). The analyses were split into three ancestry groups: Europeans, Africans, and Amerindians-Latinos. The first two groups are composed of individuals passing the 75% threshold in their respective ancestry. The Amerindian-Latinos includes individuals in the Amerindians ancestry group (75% cut-off), and individuals in the Admixed group with at least 15% Amerindians and who identified as Hispanic/Latinos ethnicity. The rationale to include these additional individuals is to compensate the paucity of the Amerindians only group and to have a similar ancestry composition as in the Latin American Research Consortium on the Genetics of Parkinson’s Disease (LARGE-PD). Last, enriching for Amerindians ancestry enables us to assess the effect of HLA-DRB1 *04:07 since DQA1 *03:01 ~DQB1 *03:02~DRB1 *04:07 is a common haplotype in this ancestry group.

[00199] Imputation. Each cohort-genotyping platform was imputed on the TOPMed imputation server per ancestry group to obtain an imputation quality (R ² ) per ancestry group. For the local-GWAS at the HLA locus we retained variants with R ² > 0.30, MAF > 1%, and present in 50% of the imputed cohorts.

[00200] The HLA -alleles and -amino-acids were imputed on platform and ancestry specific reference panels available through HIBAG or trained in-house as previously described. In the allele-level analyses, alleles with an imputation posterior probability lower than 0.5 were considered as undetermined as recommended by HIBAG developers, and only allele with carrier frequency above1% were retained for analysis. In the haplotype-level analyses, only individuals with non-missing allele genotypes were included in the haplotype level analysis. Three-locus HLA class I or class II haplotypes were determined using the haplo.em function from the R haplo. stats package. Only haplotypes with posterior probability >0.5 and a carrier frequency of >1% were included in the analysis. In the amino-acid-level analyses, HIBAG was used to convert P-coded alleles to amino acid sequences for exon 2, 3 of HLA class I genes, and exon 2 of class II genes.

[00201] Samples retained for analysis. FIG. 16 describes the demographics of individuals retained for analysis. Analyses were implemented into 6 different groups separating WGS data and TOPMed imputed and by ancestry group: ADSP-European, ADSP-African, ADSP- Amerindian-Latino, ADGC-European, ADGC-African, ADGC-Amerindian-Latino.

[00202] Statistical analyses. In the following paragraph a variable refers indifferently to a variant in the local-GWAS at HLA locus, an HLA-allele, an HLA-haplotype, or an HLA-amino- acids. The AD risk associated with each variable was estimated using a linear mixed model regression on case-control diagnosis. The HLA -allele, -haplotype, and -amino-acids level analyses were run as dominant model (collapsing homozygotes for the minor frequency variable with heterozygotes). All statistical analyses were performed in R (v4.0.2) and adjusted for sex, six genetic principal components estimated with the PC-Air method implemented in GENESIS, and covaried by a sparse genetic relationship matrix estimated with the PC-Relate method implemented in GENESIS. Case-control analyses were not adjusted for age given that controls were older than cases in some subgroups. Correcting for age when cases are younger than controls leads to the model incorrectly inferring the age effect on AD risk, resulting in statistical power loss. [00203] Alzheimer’s Disease - UK Biobank dataset. Participants, quality control and variant imputation. The UK Biobank data includes 488,377 participants which were genotyped on single nucleotide polymorphism (SNP) microarrays and imputed at high resolution using two reference panels: (i) the Haplotype Reference Consortium (HRC) for most variants with minor allele frequency > 0.001 and (ii) the UK10K+1 OOOGenomes for variants not in the HRC panel. The quality control prior to imputation has been extensively described in Bycroft et al. The proxy-AD phenotype defined in Bellenguez et al. (i.e., cases are individuals who have an ICD10 code linked to AD in their medical record or reported a first degree with Alzheimer’s disease, March, 2021 release). We restricted our analysis to 388,051 unrelated individuals after pruning for 3 ^rd degree relatedness using the following criteria to rank order individuals for removal: (i) highest number of relatives, (ii) not proxy-AD cases (iii) and youngest individual.

[00204] Ancestry determination. We split the UK Biobank unrelated individuals into two groups: British ancestry and non-British/other ancestries. The British group correspond to identified who self-identified as white British and who clustered on together in the principal ancestry component analysis performed in Bycroft et al. (field ID: 22006). The British ancestry group was composed of 52,426 proxy-AD cases, and 272,624 controls. The non-British/other ancestries group was composed of 7,840 proxy-AD cases and 55,161 controls. This last group was heterogeneous in term of ancestral origin, but the majority of individuals identified as nonBritish European.

[00205] HLA Imputation. The HLA -alleles and -amino-acids were imputed on platform and ancestry specific reference panels available through HI BAG or trained in-house as previously described. In the allele-level analyses, alleles with an imputation posterior probability lower than 0.5 were considered as undetermined as recommended by HIBAG developers, and only allele with carrier frequency above1% were retained for analysis. In the haplotype-level analyses, only individuals with non-missing allele genotypes were included in the haplotype level analysis. Three-locus HLA class I or class II haplotypes were determined using the haplo.em function from the R haplo. stats package. Only haplotypes with posterior probability >0.5 and a carrier frequency of >1% were included in the analysis. In the amino-acid-level analyses, HIBAG was used to convert P-coded alleles to amino acid sequences for exon 2, 3 of HLA class I genes, and exon 2 of class II genes.

[00206] Statistical analyses. In the following paragraph a variable refers indifferently to a variant in the local-GWAS at HLA locus, an HLA-allele, an HLA-haplotype, or an HLA-amino- acids. The HLA -allele, -haplotype, and -amino-acids level analyses were run as dominant model (collapsing homozygotes for the minor frequency variable with heterozygotes). Proxy- AD association were tested with plink2 (v2.00a2LM) using the -glm flag covarying for age at last visit, sex, genotyping array, assessment center and the first 20 PCs provided by the UK Biobank.

[00207] Alzheimer’s Disease Neuropathology - NACC and RUSH datasets. Participants and sources of data. Participants were enrolled and followed up at one of Alzheimer’s Disease Center (ADC) across the US. Genetic data were obtained from the Rush Religious Orders Study and Memory and Aging Project (ROSMAP) and from the Alzheimer’s Disease Center (ADC) cohorts 1 to 7 parts of the ADGC (see FIG. 14 for data accession number). ROSMAP samples were assessed by the Rush ADC and their neuropathological assessment followed procedures described respectively in. Neuropathological assessment for samples with genotyping from ADGC was obtained from National Alzheimer’s Coordinating Center (NACC) and followed postmortem evaluation protocol.

[00208] Quality control procedures, ancestry determination, and imputation. The content of this section is identical to the corresponding sections in “Alzheimer’s Disease - ADSP & ADGC datasets” given that these samples were included in the association with AD status.

[00209] Samples retained for analysis. FIG. 17 describes the demographics of individuals retained for neuropathology analyses: tau Braak staging, neuritic plaques density. We also defined three categories: AD pathology only, Lewy body (LB) pathology only, and dual pathology (AD and LB) and compared these against controls without AD and LB pathologies. FIG. 19 describes these categories and follows the classification defined in.

[00210] FIG. 18 provides the demographics and number of individuals per category.

[00211] Statistical analyses. The statistical analyses follow the method described in the “Alzheimer’s Disease - ADSP & ADGC datasets” corresponding section.

Example 2

HLA-DR4 meditated resilience to neurodegeneration

[00212] HLA-DR4 presentation of ac-PHF6 to T cells to protect against Tau spreading/aggregation is assessed by: 1 ) vaccinating DR4-transgenic mice with ac-PHF6 and monitoring Tau spreading in vivo. 2) isolating and characterizing ac-PHF6-autoreactive T cells in humans using single cell sequencing and expression in Jurkat cells. 3) Determining frequency of ac-PHF6- reactive T cell and corelating responses with disease evolution.

[00213] Our genetic studies in Example 1 show the presence of a protective immune response in specific individuals carrying the DR4 allele. These subjects can mount a response against a particular piece of Tau central to aggregation (ac-PHF6). It is believed that an HLA- DRB1 *04-mediated adaptive immune response decreases PD and AD risk, by acting against ac-PHF6, a critical element of Tau seeding. PHF6 is not only an integral portion of all known Tau aggregate structures, it is also necessary for RT-Quic of R4 containing Tau. Because the ac-PHF6 T cell responses is expected to be protective, an increased ac-PHF6 effector response should be associated with slower/delayed progression whereas responses against the two other Tau peptides will be uncorrelated.

[00214] Vaccinating DR4 subjects (30% of the population) with ac-PHF6, or administering antibodies targeting ac-PHF6 could protect or delay the onset of AD and PD. It is tested whether DR4-immune mediated response can slow down Tau spreading in an established mouse model. Vaccinating DR4 transgenic mice with ac-PHF6 is performed to demonstrate that an immune response directed against this epitope develops and can slow down Tau spreading in vivo. Tau spreading is tested following seeding of mice brain with human Tau extract or ac-PHF6.

[00215] A DRB1 *04:01 transgenic mice (also knock out for I A, equivalent to HLA-DR) is available at Taconic. The HLA-DR4 molecules in these mice contains mouse l-E (equivalent HLA-DQ), with mice a2 and [32 domains of lAs (equivalent to DR) and human DR4 to ensure maintained interaction of DRB1 with the mouse CD4 co-receptor. The model has been used in the study of rheumatoid arthritis, also associated with DRB1 *04:01 (RA is known to be protective of PD). The model has been used for isolation of DR4-specific T cells reactive to PTM-modified citrullinated-fibronectin (RA autoantigen) following peptide vaccination, an equivalent to the ac-PHF6 studies.

[00216] Mice models used in the study of Tau spreading are numerous, some using the P301 S hTau mutation found in FTDP or S19, P301 L, and rTg4510 transgenic mice, other using AAV- Tau of mutated Tau species. The oldest model is injecting sarkosyl-insoluble Tau extracts of AD patients into one hemisphere and waiting 1-3 months for progression of the Taupathy to the contralateral side (monitored through Tau staining). Other tau sequences containing PHF6 could be used as seeds.

[00217] The Tau spreading model is established using wild type mice first. The PHF6 binder sequence is identical in mice and acetylated at the same residue, although cryo-EM structures of the corresponding mice Tau aggregates have not yet been examined in these models It is also tested if Tau spreading can be induced by the PHF6 sequence only, with and without acetylated 311 K. Sequences used will be SEQ ID NO:12 SVQIVY(ac-K)PVDLS-NH2 and SEQ ID NO:13 SVQIVYKPVDLS-NH2). DR4 mice are vaccinated with acetylated PHF6, wild type PHF6 (a control) or saline, and after one week, injected with Tau AD extracts (and the ac- PHF6 and PHF6 peptides if these induce spreading), to examine Tau spreading 1 -3 months later. Local white blood cell infiltration at the level of the injection is examined and characterized. About 20 DR4 transgenic mice, 20 congenic animals, and 20 wild type mice are used. In selected cases or additional mice, spleens and cervical lymph nodes are dissected to collect T cells that are likely extremely enriched in reactivity to ac-PHF6 (approximately 2 weeks after ac-PHF6 vaccination). These T cells are isolated using DR4 tetramers and their TCR sequenced. IEDB MHC class II prediction program does not predict any binding for SEQ ID NO:12 SVQIVY(ac-K)PVDLS or SEQ ID NO:13 SVQIVYKVDLS to mouse IE (rank 105 and 60) so the corresponding congenic mice should not be able to mount any immune response against ac-PHF6 in the absence of DR4 and the DR4 transgenic mice should only mount a Tau DR4 restricted response against SVQIVY(ac-K)PVDLS.

[00218] It is expected that in DR4 transgenic, Tau spreading following PHF AD extracts (and perhaps ac-PHF6 brain injections) will be delayed vs congenic, independent of vaccination. With ac-PHF6 vaccination (and not PHF6), Tau spreading will be further slowed down in DR4 transgenic mice.

[00219] Ac-PHF6 autoreactive T cells are isolated and characterized through single cell sequencing (with T cell receptors). This will confirm that acPHF6 reactive T cells are effector and not regulatory T cells, thus likely to slow down disease. Preliminary data indicate that T cells recognizing ac-PHF6 presented by DRB1 *04:01 and DRB1 *04:04 exist, with significant differences in frequency across individuals. Tau 5-19 (Tau-2, N-terminal end) and 421-435 (Tau-446, C terminal end), two other peptides binding DRB1 *04:01 and DRB1 *04:04 but not DRB1 *04:05 are used as controls.

[00220] PBMCs samples from DRB1 *04:01 and DRB1 *04:04 patients with AD, PD and controls, are used, with an initial primary focus on three DR4 controls (two DRB1 *04:01 and one DRB1 *04:04) for whom we have collected leuko-pheresis and thus have a very large number of PBMCs (several billions). These controls are used to set up protocols: 1) using tetramer-linked magnetic beads for enrichment of cells recognizing ac-PHF6, Tau 5-19 (Tau- 2, N-terminal end) and Tau 421-435 (Tau-446, C terminal end) presented by DRB1 *04:01 and DRB1 *04:04 without culture and 2) tetramer isolation of the same cells following 7 days cell culture in the presence of ac-PHF6, Tau 5-19 (Tau-2, N-terminal end) and Tau-421 -435 (Tau 446, C terminal end) to enrich target cells; comparison of 1 ) and 2) will ensure cultures do not alter phenotype.

[00221] Cells binding the corresponding DR4 tetramers (labelled through 3 colors, Figure 5) will be isolated by FACS in 96 well plates. Following this, each well/cell will be amplified for total transcriptomics and TCR cx/p amplification as done in narcolepsy. Total transcriptomic profile (UMAP) is used to characterize the type of T cell isolated (expected clusters: regulatory T cell-inhibitory of the immune process, effector T cell likely to protect against the disease, or exhausted T cell representative of excessive antigenic stimulation). Experiments using tetramer-linked beads are used to demonstrate that a 7-day antigenic culture with the Tau peptides does not change the phenotype of the T cells, as measured by transcriptomics. The same protocol is used to isolate and characterize mice T cells reactive to ac-PHF6 following vaccination with ac-PHF6. [00222] It is expected that T cells recognizing ac PHF6 when presented by DR4 will be effector T cells in healthy control subjects. For cells recognizing Tau 5-19 or 421 -435, an effector or T reg phenotype is possible as these sequences do not need to be post-translationally modified to bind DR4, and as such may have been negatively selected by the thymus. We expect a large diversity of TCR sequences recognizing ac-PHF6 bound to DR4 (same for other epitopes if such cells are found to exist). However, specific sequence motives will be identified that allow binding to ac-PHF6 presented by DR4 with TCR activation. This “anticipated TCR sequence space” recognizing ac-PHF6 will be used to seek if similar sequences are found in T cell isolated from the CSF of PD or AD patients versus controls when carrying HLA- DR4.

[00223] Frequency and evolution of ac-PHF6 reactive T cells longitudinally in AD and PD, is determined correlating the immune response with evolution. As control, reactivity against Tau 5-19 (N terminal end) and 421 -435 (C terminal end) is tested, other two peptides binding DRB1 *04:01 and DRB1 *04:04. It is expected that an increased ac-PHF6 response will be associated with slower/delayed progression whereas responses against other Tau epitopes binding DR4 will be uncorrelated.

[00224] DR4 MCl/old controls and REM Behavior Disorder (RBD) patient samples will be tested for the presence and frequency of ac-PHF6 and Tau 421 -435 reactive T cells using the corresponding tetramer. Follow up samples 4-5 years later are tested, with the hypothesis that samples with the highest baseline T cell reactivity against ac PHF6 but not Tau 5-19 or 421 - 435 will have converted less to AD or PD.

Example 3

Specific epitopes to CD4 ⁺ T cell receptors (TCRs)

[00225] To identify and characterize AD-associated T cells activated by tau, with a primary focus on CD4 ⁺ T cells. Understanding adaptive immune mechanisms underlying AD has a high impact by contributing to the development of early blood diagnosis, offering effective ways of slowing down AD or stopping its progression and new therapeutics like chimeric antigen receptor (CAR) T-cell therapy. Our model suggests a classic CD4 ⁺ T cell help clearance of tau and A|342 through presentation of specific fragments of these proteins, with HLA-DRB1 *04 and other HLA subtypes to CD4 ⁺ T cells as the preferred mechanism. CD4 ⁺ T cells would then coordinate CD8 ⁺ T and B cell responses toward these antigens. Our systematic approach for epitope discovery uses a combinatorial panel of tetramers, demonstration of immune response, and identification of TCRs and phenotyping of the CD4 ⁺ T cell involved.

[00226] Using a library of overlapping peptides encompassing tau, with and without known post-translational modifications (PTMs), peptide binders of DRB1 *04:04 and DRB1 *04:01 (out-competing 50% of a known biotinylated peptide binder) that are unable to bind DRB1 *04:05 (collectively as DR401/4-binders) are determined. This mirrors the results of the GWAS-based association. Tau overlapping peptides (15-mer in length and 11 -mer overlapping) are screened against DRB1 *04:04, DRB1 *04:01 , DRB1 *04:03, DRB1 *04:05, DRB1 *04:06, DRB4*01 :03, DRB1 *07:01 , DQA1 *03:01 -DQB1 *03:02,

DQA1 *01 :01 ~DQB1 *05:01 and DQA1 *02:01 -DQB1 *03:03. DRB1 *07:01 is used as a negative control as it has no effect on disease susceptibility.

[00227] Neurofibrillary tangles (NFTs), present in the brain of AD and other related neurodegenerative diseases, are constituted of the microtube-associated protein tau in hyperphosphorylated and aggregated form. There are six isoforms of tau due to alternative splicing of the tau pre-mRNA and the largest one (2N4R) in human brain contains a total of 441 amino acids in length. Tau plays key roles in regulating microtube dynamics and neurite outgrowth and undergoes multiple PTMs at more than 50 sites including phosphorylation and acetylation. Hyperphosphorylated tau is detached from microtubes and then undergoes structural transition and misfolding and other modifications, further aggregates into paired helical filaments (PHFs), straight filaments (SFs) and/or NFTs. One important nucleating sequence, SEQ ID NO:14 PHF6 has been identified to strongly associate with tau aggregation.

[00228] A key role for adaptive immunity in AD and other neurodegenerative diseases has been outlined through genetic and immunological studies. A cornerstone of the adaptive immune response is the highly polymorphic HLA system, in which HLAs bind peptides derived from foreign or self-antigens, allowing recognition by T cells and subsequent coordination of immune responses. In Example 1 , a large-cohort GWAS analysis identified a novel genetic association of AD (rs601945) to hierarchical protective effects of HLA-DRB1 *04 subtypes, strongest with DRB1 *04:04, intermediary with DRB1 *04:01 and DRB1 *04:07, and absent for DRB1 *04:05, as well as association to decreased NFTs in postmortem brains and tau levels in the cerebrospinal fluid.

[00229] Together, these results suggest specific epitopes of tau presented by HLA, notably HLA-DR4, are involved in the pathophysiology of AD. A competition binding strategy is used to screen tau and A|342 peptide library for binding to DRB1 *04 subtypes and other associated HLA alleles. Testing of additional alleles is performed on other subtypes that are showing additional genetic associations (DQA1 *01 :01 -DQB1 *05:01 , DQA1 *02:01 -DQB1 *03:03).

[00230] Screening tau overlapping peptides (15-mer in length and 11-mer overlapping) initially against DRB1 *04:04, DRB 1*04:01 and DRB1 *04:05 at a high concentration using a well- established competition binding protocol. Whether a specific sequence, especially a sequence that includes a PTM (this would reduce the probability of central tolerance), binds to specific HLA alleles can be determined by in vitro screening of peptide libraries containing 11 -mer overlapping 15-mer peptides covering the entire tau and A|342 proteins, with or without known combinations of available PTMs. A summary list of peptides to be tested, originating from tau and A|342, is reported in Table 1 .

Table 1 .

Summary of main tau = peptides including any combination of PTMs. p, phosphorylation. Acetyl, acetylation, m, methylation. Ubi, ubiquitination. CIT, citrullination. NOTyr, nitration of tyrosine. MetO, oxidation of Methionine.

Total

No. PTMs w/oPTM PTM

Tau 1046 pSer, pThr, pTyr, Acetyl, mLys, mArg, pLys, Ubi-Lys 142 904

Tested

No. PTMs w/oPTM PTM

Tau 448 pSer, pThr, pTyr, Acetyl, mLys 142 306

[00231 ] Testing dose response of tau DR401/4-binders including two more DR alleles: DRB1 *04:03 and DRB1 *04:06. To examine binding affinity for these peptides, DR401/4- binders are identified and tested for binding affinity at molar ratios of 1 , 2.5, 5, 10, 20, 40, 100, 200, and 400 against five key DRB1 *04 alleles, namely DRB1 *04:04, DRB1 *04:01 , DRB1 *04:05, DRB1 *04:03 and DRB1 *04:06.

[00232] Binding affinity of tau DR401/4-binders is tested against other neurodegeneration associated HLA alleles, including DRB4*01 :03, DRB1 *07:01 , DQA1 *03:01 -DQB1 *03:02, DQA1 *01 :01 -DQB1 *05:01 and DQA1 *02:01 -DQB1 *03:03, in which DRB1 *07:01 will be used as a control tetramer. Libraries are screened for binding to other associated HLA alleles. These will include a) DRB4*01 :03 and DQA1 *03:01 -DQB1 *03:02, allelic combinations associated with DRB1 *04, and that could thus also play a role and b) DQA1 *02:01 -DQB1 *03:03 and DQA1 *01 :01 ~DQB1 *05:01 (allelic combinations associated with increased predisposition to AD/PD, although effects are weaker than protective effects of DRB1 *04). DRB1 *07:01 is screened as a control HLA subtype.

[00233] We initially focused our efforts on DRB1 *04:04, DRB1 *04:01 , and DRB1 *04:05 because of their clear hierarchical genetic associations in AD/PD. Peptides are screened for binding using an established peptide competition assay, in which each tau/A|342 peptide and known biotinylated binder peptides (bio-GAD, LPRLIAFTSEHSHFS, for DRB1 *04:04, DRB1 *04:01 , DRB1 *04:05, DRB1 *04:06 and DRB4*01 :03; bio-EBV ₄₉ o-503, SEQ ID NO:15 GLYRALLARSHVERTTDEY, for DRB1 *04:03) at a molar ratio of 800:1 are incubated with the corresponding HLA-DR in a 96-well plate pre-coated with anti-DR antibody (clone LN3), followed by time-resolved fluorescence (TRF) development. Non-specific binding is removed by extensive wash. Peptide binding is qualified based on the ability to displace bio- GAD/EBV490-503 in vitro. Peptides that out-competing Bio-GAD/ EBV490-503 by >75% are considered strong binders (SB); those with 50-75% displacement weak binders (WB).

[00234] We first verified binding affinities of anti-DR antibody (clone LN3) and bio-GAD/EBV ₄ go- 503 to culprit DR alleles. Next, we synthesized and screened 448 tau peptides originating from the longest isoform of tau (2N4R). These peptides were designed to include as many combinations as possible of the most frequent PTMs (306, all of frequency >5% and all >15% in combinations) as we could. Looking at tau DR401/4-binders that strongly or weakly bound DRB1 *04:04 and DRB1 *04:01 (<50% of bio-GAD binding) but not DRB1 *04:05, we detected four peptide regions of interest: Tau-2 SEQ ID NO:16 (5-RQEFEVMEDHAGTYG-19), Tau- 324 SEQ ID NO:17 (305-SVQIVYK[Acetyl]PVDLSKVT-319) only with acetylated K311 , Tau- 283 SEQ ID NO:18 (404-SPRHLSNVSSTGSID-418) and Tau-446/447 SEQ ID NO:19 (421 - DSPQLATLADEVSAS-335) with or without phosphorylated S422. Of particular interest, Tau- 324 contains the canonical PHF6 hexapeptide sequence SQ ID NO:14 (VQIVYK) known to be important for tau aggregation, and this region only binds DRB1 *04 when acetylated at K311 , the most frequent PTM reported in AD brains. Except for Tau-283, Tau-2, 324, 446 and 447 strongly bound DRB1 *04:04 and DRB1 *04:01 but not DRB1 *04:05 at as low molar ratio as 40:1 in a dose response test and was replicated twice.

[00235] Peptide binding prediction suggests that the PHF6 area also binds DRB4*01 :03 and, weakly DQA1 *03:01 -DQB1 *03:02 and DQA1*01 :01 -DQB1 *05:01 . For this reason, we tested binding of 44 tau DR401/4-binders against DRB4*01 :03, finding these sequences to also bind DRB4, although in this case the PTM modified acetylated peptide bound less strongly the DRB1 *04 subtypes associated with AD. Notably, non-modified PHF6 sequence containing peptide Tau-322 SEQ ID NQ:20 (305-SVQIVYKPVDLSKVT-319) strongly bound to DRB4*01 :03, while not binding to DRB1 *04:04 or DRB1 *04:01 was observed. Strong binding of the non-acetylated form may result in tolerance toward the epitope when presented by DRB4, explaining DRB4 by itself is not associated with protection. Of note, PHF6 peptides did not bind to DQA1 *03:01 -DQB1 *03:02 and DQA1 *01 :01 -DQB1 *05:01 , thus slight predisposing effect of these subtypes (Table 1) must be mediated by another mechanism.

[00236] Differential immune responses of tau between AD patients and controls using tetramer

DRB1 *04:04 and DRB1 *04:01 associated with cognate peptides. Peripheral blood mononuclear cells (PBMCs) cells from AD patients and controls are stimulated with a pool of tau cognate peptides, and stained with a panel of DRB1 *04:04 and DRB1 *04:01 -tau tetramers labelled with distinct fluorophore to measure frequency of the corresponding CD4+ T cells using fluorescence-activated cell sorting (FACS). The DRB1 *04 reactivities are likely to primarily target tau and play a neuroprotective role. [00237] In the CSF, HLA-DRB1 *04 is also associated with decreased tau levels. A parsimonious explanation may be that a critical autoantigen sequence, tau PHF6 being the best candidate, binds to HLA-DRB1 *04 and is next presented to TCRs. This initiates an immune response that reducing tau spreading (PHF6 is part of the core aggregating sequences, and acetylation of the sequence at K311 promotes aggregation. Autoimmunity directed toward PTM modified antigens is well established in multiple autoimmune diseases (e.g., celiac disease with gluten, rheumatoid arthritis with citrullinated peptides, type 1 narcolepsy with hypocretin). The development of fluorochrome-conjugated peptide-HLA (pHLA) multimers (including tetramers and dextramers) in conjunction with continuing advances in FACS has transformed the study of antigen specific T cells by enabling their visualization, enumeration, phenotypic characterization and isolation from ex vivo samples, thus HLA tetramers are an essential tool for characterizing antigen specific CD4 ⁺ T cells. We start with the premise that the immune response will be qualitatively different in DRB1 *04:04 and/or DRB1 *04:01 positive healthy vs. AD patients over time. Using tetramer DRB1 *04:04 and DRB1 *04:01 , T cells are identified that are only present/activated in the context of DRB1 *04:04 and DRB1 *04:01. In parallel with this, T cell reactivity directed against tau peptides presented by DRB4*01 :03, DQA1 *03:01 -DQB1 *03:02, DQA1 *02:01 -DQB1 *03:03, DQA1 *01 :01 ~DQB1 *05:01 and DRB1 *07:01 is determined.

[00238] HLA tetramers binding tau peptides are used, including N-terminal Tau-2 and C- terminal Tau-446/447, and peptides containing PHF6. Experiments use PBMC samples from a) 50 AD and 50 PD patients carrying DRB1 *04:04 and DRB1 *04:01 , and b) 50 age-matched controls carrying DRB1 *04:04 and DRB1 *04:01 ; c) and also include longitudinal samples in 10 AD patients, 10 PD patients and 10 healthy controls from a) and b).

[00239] As aggregation must be excluded when tetramers are generated and peptides containing the PHF6 hexasequence potentially aggregate in vitro, we have investigated PHF6 containing peptides with different length and PTMs for tetramer generation. We also examined those strong binders of PHF6 in tetramer generation process with additional size exclusion column and found soluble tetramers with a) a 13-mer Tau-527 (306- VQIVYK[Acetyl]PVDLSKV-318) with acetylated K311 for both DRB1 *04:04 and DRB1 *04:01 , b) a 15-mer Tau-449 SEQ ID NO:21 (305- SVQIVYK[Acetyl]PVDLSK[Acetyl]VT-319) with double acetylated K311 and K317 for both DRB1 *04:04 and DRB1 *04:01 and c) a 14-mer Tau-536 SEQ ID NO:22 (305-SVQIVYK[Acetyl]PVDLSK[Acetyl]V-318) with double acetylated K311 and K317 for DRB1 *04:01 only. Including the other two peptides, we decided to stimulate PMBCs with a pool of Tau-449, Tau-536, Tau-527, Tau-2 and Tau-446 and correspondingly conjugate a) R-phycoerythrin (PE) to Tau-449 and Tau-536, b) Brilliant Violet™ (BV) 421 to Tau-527, c) BV605 to Tau-446 and d) BV650 to Tau-2. [00240] Cell cultures are harvested and incubated with the combinatorial tetramer panel. Samples are analyzed by FACS and tetramer positive CD4 ⁺ T cells sorted. Human T cell markers including AF488-anti-CD4, AF700-anti-CD8, and allophycocyanin (APC)-cyanine (Cy) 7 anti-CD3 are used. As cell quality is critical for successful sequencing of T cell receptors (TCR) and whole transcriptome amplification, one AD patient sample and one control sample is processed at a time and cells kept on ice as little as possible.

[00241] To demonstrate that a DRB1 *04-restricted adaptive immune response directed against acetylated PHF6 tau is protective in AD, we tested individuals with AD, Parkinson’s disease (PD), mild cognitive impairment (MCI) and cognitively healthy age-matched controls for the presence of T cells recognizing tau peptides, including the K311 acetylated epitope presented by DRB1 *04:04 and DRB1 *04:01 .

[00242] Tetramers with PHF6 peptides. This was done using PBMCs cultured for 10 days with K311 -acetylated (K311Ac) epitopes, followed by FACS staining using DRB1 *04:01 and DRB1 *04:04 tetramers in subjects positive for these HLAs. Although DRB1 *04:01 and DRB1 *04:04 have similar affinity for this epitope in vitro, a larger number of T cells recognizing this epitope was observed in DRB1 *04:04 compared to DRB1 *04:01 subjects (p = 0.003), without clear differences in patients versus controls. In two DRB1 *04:04 individuals, one AD and one control, response was particularly striking, with sensitivity analysis showing that the difference between DRB1 *04:04 and DRB1 *04:01 remained significant when excluding these (p = 0.01 ). This assay demonstrates the existence of a DRB1 *04-acetylated PHF6 restricted immune response in vivo. Of note, two MCI samples with longitudinal status showed a slight decreased number of PHF6-restricted CD4 ⁺ T cells when developing Lewy body dementia (LBD) and AD respectively.

[00243] To address the issue of comparing responses toward multiple epitopes within a single subject, combinatorial tetramers are used. This is done using PBMCs cultured for 10 days with a pool of tau peptides, followed by FACS staining using a combination of DRB1 *04:01 and DRB1 *04:04 tetramers in subjects respectively positive for these HLAs. The advantage of this approach is that we will be able to use tetramers for single acetylated K311 PHF6, double acetylated K311 and K317 PHF6, and Tau-446 for both DRB1 *04:01 and DRB1 *04:04.

[00244] Use of tetramers loaded with PHF6 peptides and of combinatorial tetramers demonstrate that tau specific CD4 ⁺ T cells are present in AD and PD. These results are extended to more samples, including in a longitudinal context. We expect regular T cells to decrease and regulatory T cells to increase in frequency as AD and PD progresses versus controls. We also expect that relative to other DRB1 *04-restricted responses in these patients, the responses to acetylated tau will be higher in age-matched controls versus AD. To test, combinatorial tetramer experiments are extended by testing additional controls and AD patients. [00245] Characterizing T cell receptors (T CRs) and phenotyping of tau and A/342 specific CD4+

T cells from AD patients and controls. TCRs located onto CD4 ⁺ T cells are those recognizing antigens presented by HLA class II molecules such as HLA-DR and DQ. They are responsible for antigen recognition and subsequent immune responses. TCR diversity is a challenge, as a human body is estimated to carry ~10 ¹³ different clonotypes, with sequence diversity reaching up to 10 ¹⁵ and 10 ²⁰ . Human CD4 ⁺ T cells in particular, are critical regulators of the immune system. These cells are heterogeneous in response and recognize a large number of different antigens, encompassing various T subsets of specialized functions. Antigen- experienced T cells are generally fixed in their phenotype (notably when terminally differentiated), although some cells retain plasticity and can acquire additional cytokine producing phenotypes upon antigenic re-stimulation. T cells reactive to tau autoantigens such as Tau-2, Tau-446, and PHF6 peptides (Tau-449, 527 and 536) are phenotyped. T cell phenotype differences may be critical to drive pathology.

[00246] Because antigen specific CD4 ⁺ T cells toward these antigens are rare, all cells identified are single sorted into 96-well plate using FACS. TCR sequences are recovered using a three-round nested polymerase chain reaction (PCR) protocol. Full-length transcriptome sequencing with ultimate sensitivity is conducted using SMART-Seq single cell PLUS kit (Cat# R400751 , Takara Bio) as instructed.

[00247] In details, the tau specific CD4 ⁺ T cells were sorted directly into a 96-well PCR plate preloaded with 12.5ul lysis buffer containing 3’ SMART-seq CDS Primer II A and sealed well, then immediately stored at -80°C for further processing. The SMART-seq single cell kit (Cat# 634472, Takara Bio) was used for full length complementary deoxyribonucleic acid (cDNA) synthesis and LD-PCR amplification, and the SMART-Seq single cell PLUS kit (Cat# R400751 , Takara Bio) was used for library preparation according to manufactures’ protocols. Briefly, first-strand cDNA synthesis is primed by the 3' SMART -Seq CDS Primer 11 A and using the SMART-Seq oligonucleotide for template switching (TSO) at the 5' end of the transcript. For cDNA amplification, the PCR primer amplifies the cDNA by priming to the sequences introduced by the 3' SMART-Seq CDS Primer II A and the SMART-Seq TSO. The plate was placed in a preheated thermal cycler with a heated lid and run the following program: 98°C, 10 sec, 65°C, 30 sec, 68°C, 3 min for 21 cycles. PCR-amplified cDNA is purified by immobilization on NucleoMag NGS Clean-up and Size Select beads (Cat# 744970, Takara Bio). The concentration of the amplified cDNAs were carefully determined by using Qubit dsDNA HS Assay (Qubit). Enzymatically fragmented cDNA with stem-loop adapters were ligated. Illumina-compatible libraries were then amplified and indexed with unique dual indexes (UDIs, 96-plex). An equal molar amount of the tagmented cDNA library from each sample was pooled for purification and quantification. The resulting libraries per plate were sequenced on Illumina MiSeq at Human Immune Monitoring Center (HIMC, Stanford). FASTQ files were generated using the bcl2fastq2 Conversion v2.19 tool. Each set of samples seq data was aligned and normalized using STAR2 aligner to assemble the mapped reads into human transcriptome.

[00248] TCRs of interest are cloned into a specific vector, N103, in which TCRa and TCR|3 chain are separated by a self-cleavage sequence P2A. This ensure that an equal number of TCRa and TCRp copies is produced. These chimeric TCRs are synthesized and transfected via lentivirus packing into an engineered Jurkat 76 (J76) cell line that has no endogenous TCR but possesses a luciferase reporter system driven by nuclear factor of activated T-cells (NFAT), a system that react to TCR stimulation. In parallel with this, aAPCs expressing specific HLA alleles, including DRB1 *04:04 and DRB1 *04:01 are made, using K-562 cells (obtained from ATCC® and lacking any HLA) with a similar protocol to that used for TCR transfection. In these experiments, luminescence is measured after co-culturing J76-transfected-TCR (T cell like) cell lines, K-562-HLA allele specific (aAPC) cell lines and peptide. If a peptide binds the corresponding HLA and the corresponding complex is recognized by the transfected TCR, luminescence ensues.

[00249] Identifying antigen specific CD4 ⁺ T cell subsets based on their gene expression profiling is done using a well-established program, Seurat. Briefly, all cells first go through quality control (QC) (total counts >= 50,000, genes detected >= 4,000, and frequency of mitochondrial genes (%MT) <=10% for each cell). Cells that pass QC are analyzed in a standard workflow, including normalization, log transformation, regression out total counts, %MT and cell cycle sores, and batch correction (if any). These cells are further clustered using principal component analysis (PCA) and uniform manifold approximation and projection (UMAP). Cell phenotyping and TCR clone type is analyzed.

[00250] Rapid progress in single-cell RNA sequencing (scRNA-seq) allows researchers to uncover new and potentially unexpected biological discoveries and provides many valuable insights into complex biological systems, for example, can reveal complex and rare cell populations, uncover regulatory relationships between genes, and track the trajectories of distinct cell lineages in development. 10X genomics is one of the most common techniques used for single cell droplet separation and cDNA sequencing libraries preparation for its cost efficiency with thousands of cells. However, 10X technique requires a large number of cells at start for a better and reasonable cluster and loses almost 50% input cells in the final results. Therefore, 10X technique is not appropriate for rare tetramer HLA positive CD4 ⁺ T cells.

[00251 ] Here, we performed a pilot test for the whole transcriptome sequencing and TCR amplification in two 96-well plates using kits from Takara Bio. We selected one HLA- DRB1 *04:04 AD patient and one cognitively healthy control (HC) with clearly staining pattern by tetramer DRB1 *04:04-PHF6 (K31 1 Ac). These tetramer ⁺ CD4 ⁺ T cells were single-cell sorted into each well containing lysis buffer and sequenced for both the whole transcriptome and TCR. 96/96 (100%) and 92/96 (95.8%) TCR clones were recovered from AD and HC, respectively, with reliable number of TCR sequencing counts. Among the recovered TCR clones, one AD clone (AD-TCR-1 ) and one HC clone (HC-TCR-1) was highly enriched (AD: 91/96, 94.8%; HC: 72/92, 78.3%) (Table 2). Further, 82 out of 96 (85.4%) AD and 78 out of 96 (81 .2%) control cells passed quality control (total counts >= 50,000, genes >= 4,000, %MT <= 10%) for clustering. Three clusters (0, 1 and 2) were identified. Cluster 0 was consisting of AD and control cells, whilst cluster 1 and 2 was mainly from AD and controls, where the most enriched TCR clones (AD-TCR-1 and HC-TCR-1 ) were located, respectively (Table 2). Notably, expression of both FOXP3 and CTLA4 were higher in AD than control, suggesting regulatoryphenotype of PHF6 (K311Ac) restricted cells in AD and a protective role of PHF6 (K311Ac) restricted T cells only in controls. This shows that we are able to obtain TCR and whole transcriptome analysis (WTA) sequences using single cell cDNA sorted in 96-well plate by FACS in our laboratory and provides promising results.

[00252] Our experiments are the first to isolate and characterize DR4 restricted tau reactive T cells in controls and AD patients. We anticipate finding TCR epitopes that are enriched in AD patients or controls, bind to DRB1 *04:04 or DRB1 *04:01 , and are activated by tau PHF6. DRB1 *04 restricted T cells are expected to show a difference in comparison to reactivity to other epitopes and type of T cells (Treg vs T effector) involved. We expect that TCR recognizing non PTM modified peptides will be fewer, have lower peptide affinity, be more diverse in TCR sequences, and be more likely to have a Treg phenotypes. A set of conserved TCR motifs will be found using grouping of lymphocyte interactions by paratope hotspots (GLIPH).

[00253] Method for single cell whole transcriptome amplification. The specific T cells were sorted directly to a 96-well PCR plate preloaded with 12.5ul lysis buffer containing 3’ SMART- seq CDS Primer II A and sealed well then immediately stored at -80C for further processing. The SMART-seq single cell kit (Cat. #634472, Takara Bio) was used for full length cDNA synthesis and LD-PCR amplification, and the SMART-Seq single cell PLUS kit (Cat. #R400751 , Takara Bio ) was used for library preparation according to manufactures’ protocols. Briefly, First-strand cDNA synthesis is primed by the 3' SMART-Seq CDS Primer II A and uses the SMART-Seq Oligonucleotide for template switching (TSO) at the 5' end of the transcript. For cDNA amplification, the PCR Primer amplifies the cDNA by priming to the sequences introduced by the 3' SMART-Seq CDS Primer II A and the SMART-Seq sc TSO. Place the plate in a preheated thermal cycler with a heated lid and run the following program 98°C 10 sec, 65°C 30 sec, 68°C 3 min for 21 cycles. PCR-amplified cDNA is purified by immobilization on NucleoMag NGS Clean-up and Size Select (Cat. No. 744970, Takara Bio) beads. The concentration of the amplified cDNAs were carefully determined by using Qubit dsDNA HS Assay (Qubit). Enzymatically Fragmented cDNA with stem-loop adapters were ligated. Illumina-compatible libraries were then amplified and indexed with unique dual indexes (UDIs, 96-plex). An equal molar amount of the tagmented cDNA library from each sample was pooled for purification and quantification. The resulting libraries per plate were sequenced on Illumina MiSeq at Human Immune Monitoring Center (HIMC). FASTQ files were generated using the bcl2fastq2 Conversion v2.19 tool. Each set of samples seq data was aligned and normalized using STAR2 aligner to assemble the mapped reads into mouse transcriptome Homo Sapiens/HG19 Refseq and DEseq 2 was used for global gene expression analysis. The R packages (or SURAT or other tools to be specified) were used for secondary analysis and visualization (heatmap and clustering algorithm).

[00254] Shown in Figure 28, the data are striking in that for an AD patient most of the cells isolated were Treg cells, while for the healthy control most T cells were T effector/memory.

Table 2.

TCR clones recognizing tau acetyl PHF6 bound to HLA-DRB1 *04 retrieved with single cell whole transcriptome sequencing. AD, Alzheimer’s disease. HC, healthy control

Dx CDR3b CDR3a Vb Jb Va Ja DR count

AD SEQ ID NO:24 SEQ ID NO:46 BV7-8 BJ1 -2 AV12-2 AJ49 DR404 91 *

CASSLGQAYGY CAVNKGNTGN TF QFYF

HC SEQ ID NO:28 SEQ ID NO:50 BV6-1 BJ2-7 AV8-6 AJ57 DR404 72*

CASNRPGTSYE CAVSDRGGSE QYF KLVF

PD SEQ ID NO:181 SEQ ID NO:187 BV9 BJ2-7 AV19 AJ15 DR404 3

CASSGAPGTGQ CALSVTNQAG LREQYF TALIF

AD SEQ ID NO:25 SEQ ID NO:47 BV9 BJ2-3 AV1-2 AJ20 DR404 3

CASSVASTDTQ CAVRDPVNDY YF KLSF

HC SEQ ID NO:185 SEQ ID NO:191 BV20-1 BJ1 -3 AV10 AJ20 DR401 2

CSARVSGGNTI CWSTNDYKL YF SF

PD SEQ ID NO:183 SEQ ID NO:189 BV20-1 BJ1 -2 AV38-1 AJ43 DR404 2

CSARADRNYGY CALYNNNDMR TF F

AD SEQ ID NO:184 SEQ ID NO:190 BV20-1 BJ1 -4 AV29/DV AJ33 DR401 2

CSARERGLAEK CAARDFSNYQ 5

LFF LIW

HC SEQ ID NO:29 SEQ ID NO:51 BV19 BJ2-1 AV8-4 AJ11 DR404 2

CASRMASRAYN CAVSDTNSGY EQFF STLTF

HC SEQ ID NO:30 SEQ ID NO:52 BV3-1 BJ2-1 AV22 AJ58 DR404 2

CASSQPGTGSY CAEETSGSRL NEQFF TF

HC SEQ ID NO:31 SEQ ID NO:53 BV29-1 BJ2-3 AV35 AJ13 DR404 2

CSVHSTDTQYF CAGQAYSGGY QKVTF

HC SEQ ID NO:194 SEQ ID NO:195 BV9 BJ2-1 AV12-1 AJ39 DR404 1 CASSPEAASSY CVVTVTPMTG NEQFF NMLTF

HC SEQ ID NO:196 SEQ ID NO:197 BV20-1 BJ2-7 AV8-2 AJ4 DR404 1 CSARDGAGQYF CVVSLSGGYN KLIF

AD SEQ ID NO:198 SEQ ID NO:199 BV6-5 BJ1 -1 AV10 AJ47 DR401 1 CASSLHGQGGL CVVSAMEYGN NTEAFF KLVF

AD SEQ ID N0:200 SEQ ID NO:201 BV6-5 BJ2-7 AV12-1 AJ26 DR401 1 CASRSSGRAYE CVVNSYYGQN QYF FVF

AD SEQ ID NO:202 SEQ ID NO:203 BV10-3 BJ2-7 AV12-1 AJ36 DR401 1 CAARAAGGAYE CVVNIKTGANN QYF LFF

HC SEQ ID NO:204 SEQ ID NO:205 BV19 BJ2-3 AV13-2 AJ40 DR404 1 CASLSPGASTD CVRSGTYKYIF TQYF

HC SEQ ID NO:206 SEQ ID NO:207 BV18 BJ2-7 AV12-1 AJ47 DR404 1 CASSLDRVRRG CVGLYGNKLV EQYF F

AD SEQ ID NO:208 SEQ ID NO:209 BV20-1 BJ1 -4 AV4 AJ18 DR401 1 CSARERGLAEK CLVGDNRGST LFF LGRLYF

PD SEQ ID NO:210 SEQ ID N0:21 1 BV19 BJ2-4 AV26-1 AJ52 DR401 1 CASSIRQGEAK CIVSAAGGTSY NIQYF GKLTF

PD SEQ ID NO:212 SEQ ID NO:213 BV13 BJ2-1 AV26-1 AJ31 DR404 1 CASSLAGAGLH CIVRVGDARL NEQFF MF

HC SEQ ID NO:214 SEQ ID NO:215 BV20-1 BJ1 -3 AV26-1 AJ31 DR404 1 CSATRTAATSG CIVRNNNARL NTIYF MF

HC SEQ ID NO:216 SEQ ID NO:217 BV20-1 BJ1 -6 AV26-1 AJ13 DR404 1 CSARDQYSPLH CIVGYSGGYQ F KVTF

PD SEQ ID NO:218 SEQ ID NO:219 BV5-1 BJ1 -2 AV26-2 AJ54 DR404 1 CASSWTGGAD CILYLQGAQKL GYTF VF

HC SEQ ID NO:220 SEQ ID NO:221 BV20-1 BJ2-6 AV26-2 AJ53 DR401 1 CSAREAGTGAS CILSRSGGSNY GANVLTF KLTF

PD SEQ ID NO:222 SEQ ID NO:223 BV12-3 BJ2-5 AV34 AJ54 DR404 1 CASSFLSRQET CGADAQGAQK QYF LVF

AD SEQ ID NO:224 SEQ ID NO:225 BV7-2 BJ2-7 AV8-6 AJ54 DR401 1 CASSLSVSGLY CAVTPLQGAQ EQYF KLVF

HC SEQ ID NO:226 SEQ ID NO:227 BV19 BJ2-3 AV8-6 AJ40 DR404 1 CASLSPGASTD CAVSVLMGTY TQYF KYIF

PD SEQ ID NO:228 SEQ ID NO:229 BV3-1 BJ2-7 AV8-2 AJ44 DR404 1 CASSPRSYEQY CAVSNTGTAS F KLTF

AD SEQ ID NO:230 SEQ ID NO:231 BV2 BJ2-7 AV8-2 AJ37 DR401 1 CAITLGSPYEQY CAVSGSSSNT F GKLIF

AD SEQ ID NO:232 SEQ ID NO:233 BV5-1 BJ2-3 AV8-6 AJ11 DR401 1 CASRENYGTDT CAVSGASGYS

QYF TLTF

PD SEQ ID NO:234 SEQ ID NO:235 BV3-1 BJ2-2 AV8-6 AJ17 DR404 1 CASSPRGGSGE CAVSEKTAGN LFF KLTF

PD SEQ ID NO:236 SEQ ID NO:237 BV30 BJ1 -1 AV8-6 AJ52 DR401 1 CAWSRTGTEAF CAVSEEAGGT F SYGKLTF

HC SEQ ID NO:238 SEQ ID NO:239 BV4-1 BJ1 -4 AV8-6 AJ17 DR401 1 CASSPVRGGEK CAVSEAAGNK LFF LTF

HC SEQ ID NO:240 SEQ ID NO:241 BV6-6 BJ2-1 AV12-2 AJ36 DR404 1 CASSPPGGVYE CAVRYTGANN QFF LFF

HC SEQ ID NO:242 SEQ ID NO:243 BV19 BJ1 -1 AV41 AJ33 DR404 1 CASSYQGNTEA CAVRSNYQLI FF W

HC SEQ ID NO:244 SEQ ID NO:245 BV4-1 BJ1 -3 AV36/DV AJ36 DR404 1 CASSQDTGRSS CAVREDNLFF 7 GNTIYF

HC SEQ ID NO:246 SEQ ID NO:247 BV6-1 BJ2-7 AV3 AJ24 DR404 1 CASRMGAGGT CAVRAPLGTD YEQYF SWGKLQF

PD SEQ ID NO:248 SEQ ID NO:249 BV4-2 BJ2-3 AV12-2 AJ49 DR404 1 CASSQASGTSG CAVNSQNTGN VWDTQYF QFYF

HC SEQ ID NO:250 SEQ ID NO:251 BV6-6 BJ1 -1 AV20 AJ22 DR401 1 CASSPGVMNTE CAVLLSGSAR AFF QLTF

PD SEQ ID NO: 180 SEQ ID NO: BV6-6 BJ2-7 AV12-2 AJ31 DR404 1 CASRTEYYEQY 186 F CAVIRGNNNA RLMF

PD SEQ ID NO:252 SEQ ID NO:253 BV7-2 BJ1 -3 AV36/DV AJ44 DR404 1 CASSLGGGPGN CAVINTGTASK 7 TIYF LTF

HC SEQ ID NO:254 SEQ ID NO:255 BV25-1 BJ2-1 AV36/DV AJ52 DR404 1 CASSEWQGAN CAVGMGGTSY 7 NEQFF GKLTF

HC SEQ ID NO:256 SEQ ID NO:257 BV12-3 BJ2-7 AV8-3 AJ23 DR404 1 CASSLSTSGSY CAVGLYNQGG EQYF KLIF

HC SEQ ID NO:258 SEQ ID NO:259 BV20-1 BJ2-7 AV8-3 AJ7 DR404 1 CSARDGAGQYF CAVGGNNRLA F

PD SEQ ID NO:260 SEQ ID NO:261 BV6-9 BJ2-2 AV8-3 AJ23 DR401 1 CASSGDRGNT CAVGADNQGG GELFF KLIF

PD SEQ ID NO:262 SEQ ID NO:263 BV13 BJ2-1 AV36/DV AJ34 DR404 1 CASSLAGAGLH CAVEKAPYNT 7 NEQFF DKLIF

HC SEQ ID NO:264 SEQ ID NO:265 BV19 BJ1 -1 AV17 AJ22 DR404 1 CASS I KG LG TTN CATVSSGSAR TEAFF QLTF

AD SEQ ID NO:266 SEQ ID NO:267 BV19 BJ1 -5 AV36/DV AJ56 DR401 1 CASSFSDSNQP CASGTGANSK 7

QHF LTF PD CASSGDRGNT CARYNFNKFY BV6-9 BJ2-2 AV21 AJ21 DR401 1 GELFF F

HC CASSSFTGSEQ CARSGNNRKLI BV12-3 BJ2-7 AV13-2 AJ38 DR404 1 YF W

HC SEQ ID NO:268 SEQ ID NO:269 BV14 BJ2-5 AV6 AJ5 DR404 1 CASSRNSAVET CAPFDTGRRA QYF LTF

AD SEQ ID NO:270 SEQ ID NO:271 BV3-1 BJ2-1 AV19 AJ20 DR401 1 CASSQSVAGGN CALYRSNDYK EQFF LSF

PD SEQ ID NO:272 SEQ ID NO:273 BV3-1 BJ2-2 AV16 AJ6 DR404 1 CASSPRGGSGE CALYGGGSYIP LFF TF

HC SEQ ID NO:274 SEQ ID NO:275 BV19 BJ1 -1 AV16 AJ58 DR404 1 CASSYQGNTEA CALXETSGSRL FF TF

PD SEQ ID NO:276 SEQ ID NO:277 BV18 BJ1 -3 AV9-2 AJ31 DR404 1 CASSPTGETRG CALSGDARLM NTIYF F

PD SEQ ID NO:278 SEQ ID NO:279 BV20-1 BJ2-7 AV9-2 AJ4 DR404 1 CSAVFSGGGSY CALSDRKSGG EQYF YNKLIF

HC SEQ ID NO:280 SEQ ID NO:281 BV18 BJ1 -6 AV9-2 AJ17 DR404 1 CASSPIIPLPDS CALLKAAGNKL PLHF TF

PD SEQ ID NO:282 SEQ ID NO:283 BV28 BJ2-3 AV9-2 AJ49 DR404 1 CASSLREGIRTD CALKSNTGNQ TQYF FYF

HC SEQ ID NO:284 SEQ ID NO:285 BV19 BJ1 -4 AV6 AJ10 DR404 1 CASSDNSATNE CALDTGGGNK KLFF LTF

PD SEQ ID NO:286 SEQ ID NO:287 BV1 1 -2 BJ2-1 AV25 AJ48 DR404 1 CASSRGGSYNE CAIISNFGNEK QFF LTF

PD SEQ ID NO:182 SEQ ID NO:288 BV9 BJ2-2 AV8-2 AJ47 DR404 1 CASSVVAGGGE CAGGFGKLVF LFF

PD SEQ ID NO:289 SEQ ID NO:290 BV6-1 BJ2-5 AV38-1 AJ26 DR404 1 CASSEFWQETQ CAFMKLNYGQ YF NFVF

MCI SEQ ID NO:291 SEQ ID NO:292 BV24-1 BJ2-3 AV38-1 AJ23 DR404 1 CATSQGNTDTQ CAFMKHAQGG YF KLIF

HC SEQ ID NO:293 SEQ ID NO:294 BV5-1 BJ1 -1 AV13-2 AJ44 DR404 1 CASSLRSLNTE CAETGTASKLT AFF F

PD SEQ ID NO:295 SEQ ID NO:296 BV5-6 BJ1 -4 AV13-2 AJ39 DR404 1 CASSLNQGRGE CAENMDPYNA KLFF GNMLTF

AD SEQ ID NO:297 SEQ ID NO:298 BV18 BJ2-5 AV13-2 AJ31 DR401 1 CASSPPLGKET CAEINNARLMF QYF

PD SEQ ID NO:182 SEQ ID NO:188 BV9 BJ2-2 AV13-1 AJ42 DR404 1 CASSVVAGGGE CAAWDYGGS LFF QGNLIF

AD SEQ ID NO:299 SEQ ID N0:300 BV6-2 BJ2-7 AV8-2 AJ30 DR401 1 CASRDLGHEQY CAATNRDDKII AD SEQ ID NO:301 SEQ ID NO:302 BV7-9 BJ2-5 AV13-1 AJ30 DR401 1 CASNFRDTQET CAASTRDDKII QYF F

HC SEQ ID NO:303 SEQ ID NO:304 BV10-3 BJ2-1 AV23/DV AJ40 DR404 1 CAISVGQTFNN CAASKASGTY 6 EQFF KYIF

AD SEQ ID NO:305 SEQ ID NO:306 BV6-1 BJ2-3 AV13-1 AJ22 DR401 1 CASRLAGEDTQ CAASIGSSGSA YF RQLTF

HC SEQ ID NO:307 SEQ ID NO:308 BV6-2 BJ1 -1 AV13-1 AJ44 DR404 1 CASSYTRTGLN CAASIGGTASK TEAFF LTF

PD SEQ ID NO:309 SEQ ID NO:310 BV7-2 BJ1 -3 AV23/DV AJ43 DR404 1 CASSLGGGPGN CAASDMRF 6 TIYF

PD SEQ ID N0:311 SEQ ID NO:312 BV24-1 BJ2-5 AV29/DV AJ37 DR404 1 CATSESGTGGS CAASALHRGK 5 ETQYF LIF

AD SEQ ID NO:313 SEQ ID NO:314 BV20-1 BJ2-4 AV29/DV AJ29 DR401 1 CSASPGGAGNI CAASAGSGNT 5 QYF PLVF

AD SEQ ID NO:315 SEQ ID NO:316 BV7-2 BJ1 -3 AV29/DV AJ26 DR401 1 CASSLVGPSGN CAAPLRGQNF 5 TIYF VF

HC SEQ ID NO:317 SEQ ID NO:318 BV12-3 BJ2-5 AV29/DV AJ41 DR404 1 CASRTSGGGET CAALVRTRPY 5 QYF ALNF

HC SEQ ID NO:319 SEQ ID NO:320 BV9 BJ2-1 AV23/DV AJ54 DR404 1 CASSVTTSNNE CAAKRGAQKL 6 QFF VF

AD SEQ ID NO:26 SEQ ID NO:48 BV2 BJ2-3 AV8-3 AJ42 DR404 1 CASSSSGDTDT CAVGRYGGSQ QYF GNLIF

AD SEQ ID NO:27 SEQ ID NO:49 BV24-1 BJ2-5 AV5 AJ9 DR404 1 CATSGGTSGET CAESINTGGFK QYF TIF

HC SEQ ID NO:32 SEQ ID NO:54 BV12-3 BJ1 -5 AV38- AJ49 DR404 1 CASRGGFANQP CATNTGNQFY 2DV8 QHF F

HC SEQ ID NO:33 SEQ ID NO:55 BV5-8 BJ1 -1 AV12-2 AJ45 DR404 1 CASSFGQVNTE CAVTYSGGGA AFF DGLTF

HC SEQ ID NO:34 SEQ ID NO:56 BV12-3 BJ2-3 AV8-3 AJ17 DR404 1 CASSFGWGKR CAVGPAAGNK EGADTQYF LTF

HC SEQ ID NO:35 SEQ ID NO:57 BV7-9 BJ2-6 AV8-6 AJ39 DR404 1 CASSFPRGSGA CAVSENNAGN NVLTF MLTF

HC SEQ ID NO:36 SEQ ID NO:58 BV28 BJ2-3 AV12-1 AJ26 DR404 1 CASSLGGLVTD CVVADNYGQN TQYF FVF

HC SEQ ID NO:37 SEQ ID NO:59 BV7-2 BJ2-7 AV13-1 AJ22 DR404 1 CASSLVAGSHIY CAASKGSARQ EQYF LTF

HC SEQ ID NO:38 SEQ ID NO:60 BV3-1 BJ2-5 AV20 AJ57 DR404 1 CASSPGLATLG CAVQARGSEK

TQYF LVF HC SEQ ID NO:39 SEQ ID NO:61 BV7-3 BJ2-5 AV38- AJ56 DR404 1

CASSPTGQGFE CAYRFTGANS 2DV8

TQYF KLTF

HC SEQ ID NO:40 SEQ ID NO:62 BV4-3 BJ2-1 AV8-3 AJ9 DR404 1

CASSQVQGDG CAVVGNTGGF YNEQFF KTIF

HC SEQ ID NO:41 SEQ ID NO:63 BV5-1 BJ1 -1 AV8-6 AJ57 DR404 1

CASSSDRDPGF CAVSDRGGSE F KLVF

HC SEQ ID NO:42 SEQ ID NO:64 BV28 BJ2-7 AV12-2 AJ22 DR404 1

CASSVGQTYEQ CAVTGSARQL YF TF

HC SEQ ID NO:43 SEQ ID NO:65 BV15 BJ1 -4 AV25 AJ47 DR404 1

CATSREKGAGE CAGLGGNKLV KLFF F

HC SEQ ID NO:44 SEQ ID NO:66 BV30 BJ1 -5 AV23DV AJ18 DR404 1

CAWGRTGGDQ CAADRGSTLG 6

PQHF RLYF

HC SEQ ID NO:45 SEQ ID NO:67 BV29-1 BJ2-5 AV12-3 AJ15 DR404 1

CSVAQGAGETQ CATRDAGTALI YF F

HC=healthy control, PD=Parkinson's disease, AD=Alzheimer's dementia, MCi=mild cognitive impairment

*=subjects in Figure 27-28; these cells are regulatory and fox P3 positive in AD and regular T memory in the control