Methods for treating multiple sclerosis with autologous t cells

Abstract

To provide methods for treating or preventing multiple sclerosis.SOLUTION: Disclosed is a method for treating or preventing multiple sclerosis (MS) in a subject, which comprises administering to the subject autologous cytotoxic T cells (CTLs) expressing a T cell receptor that specifically binds to an EBV peptide presented on a class I MHC.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 448,707, filed on January 20, 2017, and U.S. Provisional Patent Application No. 62 / 576,349, filed on October 24, 2017, each of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Autoimmune diseases, such as multiple sclerosis (MS), are conditions that result from an abnormal immune response against the body's own tissues. MS is characterized by the breakdown of myelin, the protective lipid sheath that surrounds nerve fibers, by the body's own immune cells.

[0003] Epstein-Barr virus (EBV), also known as human herpesvirus 4, is a ubiquitous herpesvirus. Recently, it has been shown that exposure to EBV can be predisposing or otherwise play a role in the etiology of autoimmune diseases, including MS. For example, recent studies have shown that individuals diagnosed with MS exhibit higher levels of EBV-related proteins in aggregated B cells in neural tissue than healthy individuals. It is hypothesized that an increase in EBV-infected B cells and / or an incomplete elimination of such cells may predispose an individual to multiple sclerosis.

Summary of the Invention

[0004] A method of treating MS (e.g., relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS) is provided herein, which comprises administering to a subject autologous T cells (e.g., cytotoxic T cells, i.e., CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on MHC (e.g., class I MHC). In some embodiments, the MS is primary progressive MS. In some embodiments, the method comprises improving or stabilizing the symptoms of MS in a subject by administering to the subject autologous T cells (e.g., CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. Also provided herein is a method of reducing the anti-EBV IgG level in the CSF of a subject having MS by administering to the subject autologous T cells (e.g., CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on a class I MHC molecule.

[0005] In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, or at least 20% of the CTLs express CD107a, IFNγ, TNF, or IL-2. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the CTLs express CD107a, IFNγ, TNF, and IL-2. At least 5%, at least 7%, at least 8%, at least 10%, at least 15%, at least 20%, or at least 30% of the CTLs are EBV-reactive.

[0006] In some embodiments, the EBV peptide comprises an LMP1 peptide or fragment thereof, an LMP2A peptide or fragment thereof, and / or an EBNA1 peptide or fragment thereof. In some embodiments, the EBV peptide comprises the sequences listed in Table 1. In certain aspects, provided herein is a method of treating multiple sclerosis (e.g., relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS) comprising isolating a sample containing T cells (e.g., CTLs) from a subject, generating T cells that express a T cell receptor that specifically binds to an EBV peptide presented on an MHC (e.g., class I MHC), and subsequently administering the T cells to the subject. In some embodiments, a sample containing autologous T cells (e.g., a PBMC sample) is incubated with an antigen presenting cell (APC) that presents an EBV peptide on an MHC (e.g., class I MHC), thereby inducing the proliferation of peptide-specific T cells (e.g., peptide-specific autologous CTLs) in the sample, whereby the T cells are generated. In some embodiments, the APC can be induced to present an EBV peptide by incubating it with a nucleic acid construct encoding the EBV peptide (e.g., AdE1-LMPpoly). In some embodiments, the APC can be a B cell, an antigen presenting T cell, a dendritic cell, or an artificial antigen presenting cell (e.g., a cell line expressing CD80, CD83, 41BB-L, and / or CD86, such as an aK562 cell, etc.). In some embodiments, the method further comprises analyzing the expression of CD107a, IFNγ, TNF, or IL-2 by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to the subject if at least 1%, at least 5%, at least 10%, at least 15%, or at least 20% of the expanded peptide-specific autologous CTLs express CD107a, IFNγ, TNF, or IL-2. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the peptide-specific autologous CTLs express CD107a, IFNγ, TNF, and IL-2.Peptide-specific autologous CTLs may have EBV reactivity in at least 5%, at least 7%, at least 8%, at least 10%, at least 15%, at least 20%, or at least 30% of the peptide-specific autologous CTLs.

[0007] In some embodiments, the EBV peptide comprises an LMP1 peptide or fragment thereof, an LMP2A peptide or fragment thereof, and / or an EBNA1 peptide or fragment thereof. In some embodiments, the EBV peptide comprises the sequences listed in Table 1. In some embodiments, MS is primary progressive MS.

[0008] In some embodiments, 5×10 6 , 1×10 7 , 1.5×10 7 or 2×10 7 cells (e.g., CTLs) are administered to a subject. In some embodiments, an initial dose of T cells (e.g., autologous CTLs) is administered, followed by one or more additional doses of T cells (e.g., autologous CTLs) at doses that increase stepwise, for example, during the course of treatment. In some embodiments, two or more, three or more, four or more, or five or more doses are administered. The amount of T cells (e.g., autologous CTLs) may vary between the initial dose and the additional doses. For example, a low dose may be administered first, followed by a high dose. In some embodiments, at least 1, at least 2, at least 3, at least 4 or at least 5 doses are administered to a subject. The doses may be administered weekly or biweekly. In some embodiments, the subject does not experience adverse effects as a result of the administration of T cells (e.g., autologous CTLs). In some embodiments, the method comprises administering four doses of increasingly large numbers of CTLs continuously. In some embodiments, the method comprises administering a first dose of 5×10 6 CTLs, a second dose of 1×10 7 CTLs, a third dose of 1.5×10 7 CTLs, and a fourth dose of 2×10 7 CTLs.

[0009] In some embodiments, the method further comprises obtaining a first sample of cerebrospinal fluid (CSF) from a subject, analyzing the amount of anti-EBV IgG in the CSF in the first sample (preferably before CTL administration), obtaining a second sample of CSF from the subject after a period of time (preferably after CTL administration), analyzing the relative amount of anti-EBV IgG in the CSF in the second sample, and if the amount of anti-EBV IgG in the second sample is less than that in the first sample, evaluating the efficacy of adoptive immunotherapy in a subject with multiple sclerosis by the disease being stable and / or not progressing. The period can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 3 months, 6 months, or 1 year.

[0010] In some embodiments, the subject undergoes a diagnostic test, such as an EDSS test. In some embodiments, the subject receives an EDSS score before and after T cell administration. After T cell administration, the EDSS score may remain the same or may decrease (e.g., by at least 0.5 or at least 1.0).

[0011] In some embodiments, a method for selecting a subject for adoptive immunotherapy is provided herein by obtaining a sample from the subject that contains T cells (e.g., CTLs), isolating autologous T cells, determining the EBV reactivity of the autologous T cells in the sample, and if at least a threshold percentage (%) of the autologous T cells (e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70% or 80%) are EBV reactive, selecting the subject for adoptive immunotherapy.

[0012] In one aspect, provided herein is a method of selecting a subject for adoptive immunotherapy by obtaining a sample from the subject that contains T cells (e.g., CTLs), isolating autologous T cells, determining the CD107a, IFNγ, TNF, and / or IL-2 expression of the autologous T cells, and selecting the subject for adoptive immunotherapy when at least a threshold percentage (%) (e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80%) of the autologous T cells express CD107a, IFNγ, TNF, and / or IL-2. In some embodiments, the subject has MS (e.g., relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS).

Brief Description of the Drawings

[0013]

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[0014] Summary A method for treating multiple sclerosis (e.g., relapsing - remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS) in a subject using autologous T cells (e.g., CTLs) that recognize one or more EBV epitopes (e.g., EBV epitopes disclosed herein) is provided herein. In some embodiments, the method further includes isolating a sample containing T cells from the subject, incubating the T cells with antigen - presenting cells (APCs) presenting an EBV peptide (e.g., an EBV peptide disclosed herein), and generating T cells that recognize the EBV peptide presented on MHC. Also provided herein is a method for evaluating the effect of adoptive immunotherapy in a subject with multiple sclerosis by obtaining a sample of cerebrospinal fluid (CSF) from the subject both before and after administration of the T cells and analyzing the relative amount of anti - EBV IgG in the CSF.

[0015] Definition For convenience, specific terms used herein, in the examples and in the appended claims are collected here.

[0016] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0017] As used herein, the term "administer" means to provide a pharmaceutical or composition to a subject, and includes, but is not limited to, administration by a healthcare provider and self-administration. Such agents can contain, for example, the peptides described herein, the antigen-presenting cells provided herein and / or the T cells provided herein.

[0018] The term "amino acid" shall include all natural or synthetic molecules that contain both an amino functional group and an acid functional group and can be included in polymers of natural amino acids. Exemplary amino acids include natural amino acids, their analogs, derivatives and homologs, amino acid analogs having variant side chains, and all stereoisomers of any of the foregoing.

[0019] The terms "bind" or "interact" refer to an association, which can be a stable association, for example, between two molecules, such as between a TCR and a peptide / MHC, by electrostatic interactions, hydrophobic interactions, ionic interactions and / or hydrogen bond interactions, for example, under physiological conditions.

[0020] The terms "biological sample", "tissue sample", or simply "sample" each refer to an aggregate of cells obtained from a subject's tissue. The source of the tissue sample can be fresh, frozen and / or preserved organs, solid tissues such as tissue samples, biopsies or aspirates, blood or any blood component, serum, blood, body fluids such as cerebrospinal fluid, amniotic fluid, ascites or interstitial fluid, urine, saliva, feces, tears, or cells from any point in the subject's pregnancy or development.

[0021] As used herein, the term "cytokine" refers to any secreted polypeptide molecule that affects the function of cells and regulates cell - to - cell interactions in immune, inflammatory, or hematopoietic responses. Cytokines include, but are not limited to, monokines and lymphokines, regardless of which cells produce them. For example, monokines are generally said to be produced and secreted by mononuclear cells such as macrophages and / or monocytes. However, many other cells such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epidermal keratinocytes, and B lymphocytes also produce monokines. Lymphokines are generally said to be produced by lymphocyte cells. Examples of cytokines include, but are not limited to, interleukin - 1 (IL - 1), interleukin - 2 (IL - 2), interleukin - 6 (IL - 6), interleukin - 8 (IL - 8), tumor necrosis factor alpha (TNFα), and tumor necrosis factor beta (TNFβ).

[0022] The term "epitope" means a protein determinant that can specifically bind to an antibody or TCR. Epitopes usually consist of chemically active surface groups of a molecule, such as amino acids or sugar side chains. A particular epitope can be defined by a specific sequence of amino acids to which an antibody can bind.

[0023] As used herein, the phrase "pharmaceutically acceptable" refers to agents, compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, in contact with human and animal tissues, are suitable for use without excessive toxicity, irritation, allergic response, or other problems or complications and that exhibit a reasonable benefit / risk ratio.

[0024] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle involved in carrying or transporting from one organ or part of the body to another, such as a liquid or solid filler, diluent, excipient, or material encapsulating a solvent. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include the following: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffering solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0025] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to polymeric forms of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof. A polynucleotide may have any three-dimensional structure and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, may be made before or after construction of the polymer. A polynucleotide may be further modified, for example, by conjugation to a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0026] As used herein, a therapeutic agent that "prevents" a condition refers to a compound that, when administered to a statistical sample prior to the onset of a disorder or condition, reduces the occurrence of the disorder or condition in the treated sample as compared to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition as compared to an untreated control sample.

[0027] As used herein, "specific binding" refers to the ability of a TCR to bind to a peptide presented on an MHC (e.g., class I MHC or class II MHC). Typically, a TCR has a K -4 of at least about 10 DSpecifically binds to the peptide / MHC with an affinity that is at least 10-fold less, at least 100-fold less, or at least 1000-fold less than the affinity for binding to non-specific and irrelevant peptide / MHC complexes (e.g., those containing BSA peptides or casein peptides), and binds to a predetermined antigen / binding partner with an affinity (K D as represented by).

[0028] As used herein, the term "subject" means a human or non-human animal selected for treatment or therapy.

[0029] As used herein, the phrases "therapeutically effective amount" and "effective amount" mean an amount of an agent effective to produce a desired therapeutic effect in at least a subpopulation of cells of a subject, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0030] As used herein, the term "treating" a disease in a subject or "treating" a subject having or suspected of having a disease means administering a medical treatment, e.g., administration of CTLs as described herein, to the subject such that at least one symptom of the disease is reduced or prevented from worsening.

[0031] The term "vector" refers to a means by which nucleic acids can be propagated and / or transferred between organisms, cells, or cell components. Examples of vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, and artificial chromosomes, which may or may not be able to replicate autonomously or may be integrated into the chromosome of the host cell.

[0032] Peptide In some embodiments, provided herein is a method of treating multiple sclerosis (e.g., relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS) using autologous T cells (e.g., CTLs) that express a TCR that specifically binds to a peptide comprising an EBV epitope presented on MHC (e.g., class I MHC). In some embodiments, for example, a method of generating such autologous T cells is provided herein by incubating a sample comprising T cells (i.e., autologous T cells) with an antigen-presenting cell (APC) that presents one or more of the EBV epitopes described herein (e.g., an APC that presents a peptide described herein that comprises an EBV epitope on a class I MHC complex).

[0033] In some embodiments, the peptides provided herein comprise the sequence of any EBV viral protein (e.g., the sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of any EBV protein). In some embodiments, the peptides provided herein comprise 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer contiguous amino acids of an EBV viral protein.

[0034] In some embodiments, the peptides provided herein comprise the sequence of LMP1 (e.g., the sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of LMP1). In some embodiments, the peptides provided herein comprise 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer contiguous amino acids of LMP1. An exemplary LMP1 amino acid sequence is provided below (SEQ ID NO: 1): 1 mdldlergpp gprrpprgpp lssyialall llllallfwl yiimsnwtgg allvlyafal 61 mlviiiliif ifrrdllcpl galcllllmi tlllialwnl hgqalylgiv lfifgcllvl 121 giwvyfleil wrlgatiwql lafflaffld illliialyl qqnwwtllvd llwlllflai 181 liwmyyhgqr hsdehhhdds lphpqqatdd ssnhsdsnsn egrhhllvsg agdapplcsq 241 nlgapgggpd ngpqdpdntd dngpqdpdnt ddngphdplp qdpdntddng pqdpdntddn 301 gphdplphnp sdsagndggp pnlteevenk ggdrgppsmt dggggdphlp tlllgtsgsg 361 gddddphgpv qlsyyd

[0035] In some embodiments, the peptides provided herein comprise the sequence of LMP2A (e.g., the sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of LMP2A). In some embodiments, the peptides provided herein comprise 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer consecutive amino acids of LMP2A. An exemplary LMP2A amino acid sequence is provided below (SEQ ID NO: 2): 1 mgslemvpmg agppspggdp dgddggnnsq ypsasgsdgn tptppndeer esneeppppy 61 edldwgngdr hsdyqplgnq dpslylglqh dgndglpppp ysprddssqh iyeeagrgsm 121 npvclpviva pylfwlaaia ascftasvst vvtatglals llllaavass yaaaqrkllt 181 pvtvltavvt ffaicltwri edppfnsllf allaaagglq giyvlvmlvl lilayrrrwr 241 rltvcggimf lacvlvlivd avlqlspllg avtvvsmtll llafvlwlss pgglgtlgaa 301 lltlaaalal laslilgtln lttmfllmll wtlvvllics scsscpltki llarlflyal 361 allllasali aggsilqtnf kslsstefip nlfcmllliv agilfilail tewgsgnrty 421 gpvfmclggl ltmvagavwl tvmtntllsa wiltagflif ligfalfgvi rccryccyyc 481 ltleseerpp tpyrntv

[0036] In some embodiments, the peptides provided herein include the sequence of EBNA1 (e.g., the sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of EBNA1). In some embodiments, the peptides provided herein include 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer consecutive amino acids of EBNA1. An exemplary EBNA1 amino acid sequence is provided below (SEQ ID NO: 3): 1 pffhpvgead yfeylqeggp dgepdvppga ieqgpaddpg egpstgprgq gdggrrkkgg 61 wfgkhrgqgg snpkfeniae glrvllarsh vertteegtw vagvfvyggs ktslynlrrg 121 talaipqcrl tplsrlpfgm apgpgpqpgp lresivcyfm vflqthifae vlkdaikdlv 181 mtkpaptcni kvtvcsfddg vdlppwfppm vegaaaegdd gddgdeggdg degeegqe

[0037] In some embodiments, the peptide comprises the sequences of the epitopes listed in Table 1.

[0038] [Table 1]

[0039] In some embodiments, the peptides provided herein comprise two or more EBV epitopes. In some embodiments, the peptides provided herein comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 EBV epitopes. For example, in some embodiments, the peptides provided herein comprise two or more EBV epitopes linked by a linker (e.g., a polypeptide linker).

[0040] In some embodiments, the peptide sequence comprises an EBV viral protein sequence except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) conservative sequence modifications. As used herein, the term "conservative sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the interaction between a TCR and a peptide containing the amino acid sequence presented on MHC. Such conservative modifications include amino acid substitutions, additions (e.g., addition of an amino acid to the N-terminus or C-terminus of the peptide), and deletions (e.g., deletion of an amino acid from the N-terminus or C-terminus of the peptide). Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues of the peptides described herein can be replaced with other amino acid residues from the same side chain family, and the modified peptide can be tested for retention of TCR binding using methods known in the art. Modifications can be introduced into the antibody by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0041] In some embodiments, the peptides provided herein comprise a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to an EBV viral protein sequence (e.g., the sequence of a fragment of an EBV viral protein). To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). Next, the amino acid residues at corresponding amino acid positions are compared. If a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0042] In some embodiments, the peptide is a chimeric peptide or a fusion peptide. As used herein, a "chimeric peptide" or "fusion peptide" includes a peptide having a sequence provided herein that is linked to a separate peptide having sequences that are not essentially linked. For example, the separate peptide can be fused directly via a peptide bond or indirectly via a chemical linker to the N-terminus or C-terminus of the peptide provided herein. In some embodiments, the peptide provided herein is linked to a separate peptide that comprises a separate EBV epitope. In some embodiments, the peptide provided herein is linked to a peptide that comprises an epitope from another viral disease and / or infectious disease.

[0043] The chimeric or fusion peptides provided herein can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different peptide sequences can be ligated in-frame using conventional techniques such as blunt-ending or stagger-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, complementary attachment of sticky ends if necessary, alkaline phosphatase treatment to avoid unwanted ligation, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including an automated DNA synthesizer. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that generate complementary overhangs between two contiguous gene fragments, which can then be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992). In addition, a number of expression vectors already encoding the fusion portion are commercially available.

[0044] The peptides provided herein can be isolated from cell or tissue sources by appropriate purification schemes using standard protein purification techniques, can be produced by recombinant DNA technology, and / or can be chemically synthesized using standard peptide synthesis techniques. The peptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of the nucleotides encoding the peptide(s) of the invention. Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous peptides in recombinant hosts, chemical synthesis of peptides, and in vitro translation are well known in the art and are further described in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd ed., Cold Spring Harbor, N.Y., Berger and Kimmel, Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif., Merrifield, J. (1969) J. Am. Chem. Soc. 91:501, Chaiken I. M. (1981) CRC Crit. Rev. Biochem. 11:255, Kaiser et al. (1989) Science 243:187, Merrifield, B. (1986) Science 232:342, Kent, S. B. H. (1988) Annu. Rev. Biochem. 57:957, Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which are hereby incorporated by reference herein.

[0045] In certain embodiments, nucleic acid molecules encoding the peptides described herein are provided herein. In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the nucleic acid molecule is a viral vector comprising a nucleic acid molecule described herein, for example, an adenovirus-based expression vector. In some embodiments, the vectors provided herein encode a plurality of epitopes provided herein (e.g., as a polyepitope). In some embodiments, the vectors provided herein encode at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 epitopes provided herein (e.g., the epitopes provided in Table 1).

[0046] In some embodiments, the vector is AdE1-LMPpoly. The AdE1-LMPpoly vector encodes a polyepitope of defined CTL epitopes derived from LMP1 and LMP2 fused to a Gly-Ala repeat-deficient EBNA1 sequence. The AdE1-LMPpoly vector is described, for example, in Smith et al., Cancer Research 72:1116 (2012), Duraiswamy et al., Cancer Research 64:1483-9 (2004), Smith et al., J. Immunol 117:4897-906, each of which is incorporated herein by reference.

[0047] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated to the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell and can thereby replicate with the host genome. Furthermore, certain vectors are capable of directing the expression of a gene. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In some embodiments, nucleic acids operably linked to one or more regulatory sequences (e.g., a promoter) in an expression vector are provided herein. In some embodiments, a cell transcribes the nucleic acids provided herein and thereby expresses the peptides described herein. The nucleic acid molecule can be integrated into the genome of the cell or it can be extrachromosomal.

[0048] In some embodiments, cells containing the nucleic acids described herein (e.g., nucleic acids encoding the peptides described herein) are provided herein. The cells can be, for example, prokaryotic, eukaryotic, mammalian, avian, murine, and / or human. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are APCs (e.g., antigen-presenting T cells, dendritic cells, B cells, or aK562 cells). In the present method, the nucleic acids described herein can be administered to cells, for example, as nucleic acids without delivery vehicles, in combination with delivery reagents. In some embodiments, any nucleic acid delivery method known in the art can be used in the methods described herein. Suitable delivery reagents include, but are not limited to, for example, Mirus Transit TKO lipophilic reagent, Lipofectin, Lipofectamine, Cellfectin, polycations (e.g., polylysine), atelocollagen, nanoplexes, and liposomes. In some embodiments of the methods described herein, liposomes are used to deliver nucleic acids to cells or subjects. Liposomes suitable for use in the methods described herein can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids is generally guided by factors such as the desired liposome size and the half-life of the liposomes in the bloodstream. Various methods for preparing liposomes are known, for example, as described in Szoka et al., (1980), Ann. Rev. Biophys. Bioeng. 9:467, and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which are incorporated herein by reference.

[0049] Autologous T cells By administering to a subject autologous T cells (e.g., CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on an MHC, a method of treating multiple sclerosis (e.g., relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS) is provided herein. In some embodiments, the MHC is class I MHC. In some embodiments, the MHC is class II MHC.

[0050] In some embodiments, an APC that presents a peptide described herein (e.g., a peptide comprising an LMP1, LMP2A, or EBNA1 epitope sequence) is provided herein. In some embodiments, the APC is a B cell, an antigen-presenting T cell, a dendritic cell, or an artificial antigen-presenting cell (e.g., an aK562 cell).

[0051] Dendritic cells for use in this process can be prepared by collecting PBMCs from a patient sample and adhering them to plastic. Generally, the monocyte population remains and all other cells can be washed away. Next, the adherent cell population is differentiated with IL-4 and GM-CSF to produce monocyte-derived dendritic cells. These cells can be matured by the addition of IL-1β, IL-6, PGE-1, and TNF-α (which upregulate important costimulatory molecules on the surface of dendritic cells), and then transduced with one or more of the peptides provided herein.

[0052] In some embodiments, the APC is an artificial antigen-presenting cell, such as an aK562 cell. In some embodiments, the artificial antigen-presenting cell is engineered to express CD80, CD83, 41BB-L, and / or CD86. Examples of artificial antigen-presenting cells, such as aK562 cells, are described in U.S. Patent Application Publication No. 2003 / 0147869, which is incorporated herein by reference.

[0053] In certain embodiments, provided herein is a method of generating an antigen-presenting cell (APC) that presents one or more EBV epitopes, comprising contacting a peptide comprising an EBV epitope described herein and / or a nucleic acid encoding an EBV epitope with an APC. In some embodiments, the APC is irradiated. In some embodiments, an APC that presents a peptide described herein (e.g., a peptide comprising an LMP1, LMP2A, or EBNA1 epitope sequence). Cells that present a peptide described herein can be produced by standard techniques known in the art. For example, cells can be pulsed to facilitate peptide uptake. In some embodiments, the cells are transfected with a nucleic acid encoding a peptide provided herein. Also provided herein is a method of producing an antigen-presenting cell (APC) comprising the step of pulsing the cells with a peptide described herein. Exemplary examples of producing antigen-presenting cells can be found in WO2013088114, which is incorporated herein by reference in its entirety.

[0054] In some embodiments, provided herein are T cells (e.g., CD4 T cells and / or CD8 T cells) that express a T cell receptor (e.g., an αβ TCR or a γδ TCR) that recognizes a peptide described herein presented on an MHC. In some embodiments, the T cells are CD8 T cells (e.g., CTLs) that express a TCR that recognizes a peptide described herein presented on class I MHC. In some embodiments, the T cells are CD4 T cells (e.g., helper T cells) that recognize a peptide described herein presented on class II MHC.

[0055] In some embodiments, methods are provided herein for generating, activating, and / or inducing proliferation of T cells (e.g., autologous CTLs) that recognize one or more of the EBV epitopes described herein. In some embodiments, a sample containing autologous T cells (i.e., a PBMC sample) is incubated in culture with an APC provided herein (e.g., an APC that presents a peptide containing an EBV epitope on a class I MHC complex). In some embodiments, the APC is autologous to the subject from whom the T cells are obtained. In some embodiments, the APC is not autologous to the subject from whom the T cells are obtained. In some embodiments, the sample containing T cells is incubated more than once with an APC provided herein. In some embodiments, the T cells are incubated with the APC in the presence of at least one cytokine. In some embodiments, the cytokine is IL-4, IL-7, and / or IL-15. Exemplary methods for inducing T cell proliferation using APCs are provided, for example, in U.S. Patent Application Publication No. 2015 / 0017723, which is incorporated herein by reference.

[0056] In some embodiments, compositions (e.g., therapeutic compositions) are provided herein that include the T cells and / or APCs provided herein and are used for treating and / or preventing multiple sclerosis in a subject by administering an effective amount of the composition to the subject. In some aspects, methods for treating multiple sclerosis using a composition (e.g., a pharmaceutical composition such as a composition containing autologous CTLs) are provided herein. In some embodiments, the composition includes a combination of a plurality (e.g., two or more) of the CTLs provided herein.

[0057] Treatment method In some embodiments, provided herein is a method of treating MS (e.g., primary progressive MS) in a subject by administering autologous T cells (e.g., autologous CTLs) provided herein to the subject. In some embodiments, the MS is relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS. In some embodiments, the autologous T cells are isolated from a peripheral blood mononuclear cell sample. Expression of biomarkers by the autologous T cells can be evaluated by any suitable method, such as flow cytometry. In some embodiments, the autologous T cells are stimulated with a vector (e.g., AdE1-LMPpoly) containing EBV viral peptides. In some embodiments, the autologous T cells are stimulated with a viral vector and sorted by flow cytometry. For example, the autologous T cells can be surface stained according to the protocol exemplified in Example 2. In some embodiments, the autologous T cells are incubated with one or more antibodies specific for CD107A and subsequently sorted by flow cytometry. In some embodiments, the autologous T cells are incubated with one or more antibodies that bind to intracellular cytokines, such as antibodies specific for IFNγ, IL-2, and / or TNF. In some embodiments, the autologous T cells are incubated with antibodies against intracellular cytokines and subsequently sorted by flow cytometry.

[0058] In some aspects, provided herein is a method of selecting a subject for adoptive immunotherapy by obtaining a PMBC sample from the subject, isolating autologous T cells, determining the EBV reactivity of the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells are EBV reactive.

[0059] In some embodiments, a method for selecting a subject for adoptive immunotherapy is provided herein by obtaining a sample containing T cells (e.g., CTLs) from the subject, isolating autologous T cells, determining the CD107A expression of the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells express CD107A.

[0060] In some embodiments, a method for selecting a subject for adoptive immunotherapy is provided herein by obtaining a sample containing T cells (e.g., CTLs) from the subject, isolating autologous T cells, determining the IFNγ expression of the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells express IFNγ.

[0061] In some embodiments, a method for selecting a subject for adoptive immunotherapy is provided herein by obtaining a sample containing T cells (e.g., CTLs) from the subject, isolating autologous T cells, determining the TNF expression of the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells express TNF.

[0062] In some embodiments, a method of selecting a subject for adoptive immunotherapy is provided herein by obtaining a sample from the subject that contains T cells (e.g., CTLs), isolating autologous T cells, determining the IL-2 expression of the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70% or 80% of the autologous T cells express Il-2.

[0063] In some embodiments, the method further comprises obtaining a sample from the subject that contains T cells (e.g., obtaining a PBMC sample from the subject). In some embodiments, autologous T cells (e.g., CD4+ T cells or CD8+ T cells) are isolated from the sample. In some embodiments, the sample consists mostly or entirely of autologous T cells.

[0064] Provided herein is a method of treating or preventing multiple sclerosis (MS) in a subject, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the T cells (e.g., CTLs) in a sample express CD107A. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the T cells (e.g., CTLs) in a sample express IFNγ. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the T cells (e.g., CTLs) in a sample express TNF. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the T cells (e.g., CTLs) in a sample express IL-2.

[0065] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) in the sample express CD107A and IFNγ.

[0066] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) in the sample express CD107A and TNF.

[0067] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) in the sample express CD107A and IL-2.

[0068] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) in the sample express IFNγ and TNF.

[0069] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) in the sample express IFNγ and IL-2.

[0070] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) in the sample express TNF and IL-2.

[0071] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) in the sample express IFNγ, TNF, and IL-2.

[0072] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) in the sample express CD107A, TNF, and IL-2.

[0073] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) in the sample express CD107A, IFNγ, and IL-2.

[0074] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) in the sample express CD107A, IFNγ, and TNF.

[0075] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) in the sample express CD107A, IFNγ, TNF, and IL-2.

[0076] In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the T cells (e.g., CTLs) are EBV-reactive.

[0077] The expression of the T cell biomarker and / or EBV-reactivity can be measured and / or analyzed either before or after T cell expansion (proliferation) using the nucleic acid constructs disclosed herein, the polypeptides disclosed herein, or the APCs.

[0078] In some embodiments, a method for treating or preventing MS in a subject is provided herein by incubating an antigen-presenting cell (APC) with a nucleic acid construct encoding an EBV peptide, thereby inducing the APC to present the EBV peptide and inducing the proliferation of peptide-specific T cells (e.g., CTLs), incubating a sample containing autologous T cells (e.g., CTLs) with the antigen-presenting cell (APC), thereby inducing the autologous T cells (e.g., CTLs) to proliferate, and administering to the subject the peptide-specific autologous T cells (e.g., CTLs). In some embodiments, EBV reactivity and biomarker expression are quantified before stimulating autologous T cells with a viral vector (e.g., a viral vector disclosed herein) and / or an APC (e.g., an APC disclosed herein). Alternatively or in addition, EBV reactivity and biomarker expression may be quantified after stimulating autologous T cells with a viral vector (e.g., a viral vector disclosed herein) and / or an APC (e.g., an APC transfected with a viral vector disclosed herein). In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of T cells in a sample that express CD107A. In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of T cells in a sample that express IFNγ. In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of T cells in a sample that express TNF. In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of T cells in a sample that express IL-2. In some embodiments, EBV reactivity is measured as the percentage (%) of T cells that express multiple biomarkers (e.g., two or more, preferably all four, of CD107A, IFNγ, TNF, and IL-2). In some embodiments, EBV reactivity is calculated by quantifying the percentage (%) of autologous T cells in a sample that express CD107A, IFNγ, TNF, and IL-2. The T cells can be isolated from a sample (e.g., a PBMC sample or a sample containing T cells) either before or after quantification of the percentage (%) of EBV reactivity. Thus, in some embodiments, EBV reactivity is the percentage (%) of T cells with the desired characteristics in a sample that mostly contains T cells.

[0079] In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of CD8+ lymphocytes in a sample that expresses CD107A. In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of CD8+ lymphocytes in a sample that expresses IFNγ. In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of CD8+ lymphocytes in a sample that expresses TNF. In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of CD8+ lymphocytes in a sample that expresses IL-2. In some embodiments, EBV reactivity is measured as the percentage (%) of CD8+ lymphocytes that express multiple biomarkers (e.g., two or more, preferably all four, of CD107A, IFNγ, TNF, and IL-2). CD8+ lymphocytes can be isolated from a sample (e.g., a PBMC sample or a sample of CD8+ lymphocytes), either before or after quantification of the percentage (%) of EBV reactivity. Thus, in some embodiments, EBV reactivity is the percentage (%) of CD8+ lymphocytes having a desired characteristic in a sample that mostly consists of or contains CD8+ lymphocytes.

[0080] In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of CD3+ lymphocytes in a sample that expresses CD107A. In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of CD3+ lymphocytes in a sample that expresses IFNγ. In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of CD3+ lymphocytes in a sample that expresses TNF. In some embodiments, EBV reactivity is measured by quantifying the percentage (%) of CD3+ lymphocytes in a sample that expresses IL-2. In some embodiments, EBV reactivity is measured as the percentage (%) of CD3+ lymphocytes that express multiple biomarkers (e.g., two or more, preferably all four, of CD107A, IFNγ, TNF, and IL-2). CD3+ lymphocytes can be isolated from a sample (e.g., a PBMC sample or a sample of CD3+ lymphocytes), either before or after quantification of the percentage (%) of EBV reactivity. Thus, in some embodiments, EBV reactivity is the percentage (%) of CD3+ lymphocytes having a desired characteristic in a sample that mostly comprises CD3+ lymphocytes.

[0081] In some embodiments, the method further comprises analyzing the expression of CD107a, IFNγ, TNF, or IL-2 by expanded peptide-specific autologous T cells (e.g., CTLs), and administering to a subject the expanded peptide-specific autologous T cells (e.g., CTLs) when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the expanded peptide-specific autologous T cells (e.g., CTLs) express CD107a, IFNγ, TNF, or IL-2.

[0082] In some embodiments, the method further comprises analyzing the expression of CD107a and TNF by expanded peptide-specific autologous T cells (e.g., CTLs), and administering the peptide-specific autologous T cells (e.g., CTLs) to a subject when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the expanded peptide-specific autologous T cells (e.g., CTLs) express CD107a and TNF.

[0083] In some embodiments, the method further comprises analyzing the expression of CD107a and IFNγ by expanded peptide-specific autologous T cells (e.g., CTLs), and administering to the subject the peptide-specific autologous T cells (e.g., CTLs) when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the expanded peptide-specific autologous T cells (e.g., CTLs) express CD107a and IFNγ.

[0084] In some embodiments, the method further comprises analyzing the expression of CD107a and IL-2 by expanded peptide-specific autologous T cells (e.g., CTLs), and administering the peptide-specific autologous T cells (e.g., CTLs) to a subject when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the expanded peptide-specific autologous T cells (e.g., CTLs) express CD107a and IL-2.

[0085] In some embodiments, the method further comprises analyzing the expression of TNF and IL-2 by the expanded peptide-specific autologous T cells (e.g., CTLs), and administering the peptide-specific autologous T cells (e.g., CTLs) to a subject when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the expanded peptide-specific autologous T cells (e.g., CTLs) express TNF and IL-2.

[0086] In some embodiments, the method further comprises analyzing the expression of IFNγ and IL-2 by expanded peptide-specific autologous T cells (e.g., CTLs), and administering the peptide-specific autologous T cells (e.g., CTLs) to a subject when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the expanded peptide-specific autologous T cells (e.g., CTLs) express IFNγ and IL-2.

[0087] In some embodiments, the method further comprises analyzing the expression of IFNγ and TNF by expanded peptide-specific autologous T cells (e.g., CTLs), and administering the expanded peptide-specific autologous T cells (e.g., CTLs) to a subject when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the expanded peptide-specific autologous T cells (e.g., CTLs) express IFNγ and TNF.

[0088] In some embodiments, the method further comprises analyzing the expression of CD107a, IFNγ, and TNF by expanded peptide-specific autologous T cells (e.g., CTLs), and administering the peptide-specific autologous T cells (e.g., CTLs) to a subject when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the expanded peptide-specific autologous T cells (e.g., CTLs) express CD107a, IFNγ, and TNF.

[0089] In some embodiments, the method further comprises analyzing the expression of CD107a, IFNγ, and IL-2 by expanded peptide-specific autologous T cells (e.g., CTLs), and administering the peptide-specific autologous T cells (e.g., CTLs) to a subject when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the expanded peptide-specific autologous T cells (e.g., CTLs) express CD107a, IFNγ, and IL-2.

[0090] In some embodiments, the method further comprises analyzing the expression of CD107a, IL-2, and TNF by expanded peptide-specific autologous T cells (e.g., CTLs), and administering to the subject expanded peptide-specific autologous T cells (e.g., CTLs) when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the expanded peptide-specific autologous T cells (e.g., CTLs) express CD107a, IL-2, and TNF.

[0091] In some embodiments, the method further comprises analyzing the expression of IFNγ, IL-2, and TNF by expanded peptide-specific autologous T cells (e.g., CTLs), and administering to a subject the expanded peptide-specific autologous T cells (e.g., CTLs) when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the expanded peptide-specific autologous T cells (e.g., CTLs) express IFNγ, IL-2, and TNF.

[0092] In some embodiments, when at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the autologous T cells (e.g., CTLs) express CD107a, IFNγ, TNF, and IL-2, the autologous T cells (e.g., CTLs) are administered to a subject.

[0093] Peptide-specific autologous T cells (e.g., CTLs) may have EBV reactivity at at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the peptide-specific autologous T cells (e.g., CTLs).

[0094] In some embodiments, from about 1×10 5 to about 1×10 8 T cells per dose of T cells are administered to a subject. In some embodiments, from about 1×10 6 to about 1×10 7 T cells per dose of T cells are administered to a subject. In some embodiments, 5×10 6 , 1×10 7 , 1.5×10 7 , or 2×10 7Administer to individual T cells (e.g., CTLs). Multiple doses may be administered. In some embodiments, an initial dose of T cells (e.g., autologous CTLs) is administered, and one or more additional doses of T cells (e.g., autologous CTLs) are administered at doses that increase stepwise, for example, along the course of treatment. In some embodiments, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more doses are administered. Additional doses that are the same as or different from the initial dose may be administered to the subject. For example, a low dose may be administered followed by a high dose. The doses may be administered daily, twice a week, once a week, every other week, once a month, once every two months, once every three months, or once every six months. In some embodiments, the subject does not experience adverse effects as a result of the administration of T cells (e.g., autologous CTLs).

[0095] In some embodiments, the method further comprises obtaining a first sample of cerebrospinal fluid (CSF) from a subject, analyzing the amount of anti-EBV IgG in the CSF in the first sample (preferably, before CTL administration), obtaining a second sample of CSF from the subject after a period of time (preferably, after CTL administration), analyzing the amount of anti-EBV IgG in the CSF in the second sample, and evaluating the efficacy of adoptive immunotherapy in a subject with multiple sclerosis by determining that the amount of anti-EBV IgG in the second sample is less than that in the first sample, indicating that the disease is stable and / or not progressing. Additional samples of CSF may be obtained and compared to previous samples. Also provided herein is a method of reducing anti-EBV IgG levels in the CSF of a subject with MS by administering autologous T cells (e.g., CTLs) that express a T cell receptor that specifically binds to EBV peptides presented on class I MHC. Reduction of anti-EBV IgG levels in the CSF can be measured by the CSF IgG index. In some embodiments, the CSF IgG level can be calculated by the formula of Reiber and Felgenhauer (i.e., see Figure 1). The anti-EBV IgG level can be reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% after administration of the T cells.

[0096] In some embodiments, the method includes improving or stabilizing symptoms of MS in a subject (e.g., visual loss, loss of visual acuity, loss or reduction of dexterity of the hands, increased fatigue and / or urgency of urination) by administering to the subject autologous T cells (e.g., CTLs, such as peptide-specific autologous CTLs described herein) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. Also provided herein is a method of improving vision, improving color vision, or stabilizing visual decline in a subject having MS, including administering autologous T cells (e.g., CTLs, such as peptide-specific autologous CTLs described herein) to the subject. In some embodiments, provided herein is a method of improving motor ability, balance, or dexterity of the hands in a subject having MS, including administering the autologous T cells described herein to the subject. Also provided herein is a method of improving sleep in a subject, including administering autologous T cells (e.g., CTLs, such as peptide-specific autologous CTLs described herein) to the subject.

[0097] In some embodiments, the subject undergoes a diagnostic test, such as the EDSS. In some embodiments, the subject undergoes an EDSS test and receives an EDSS score before and / or after T cell administration. After T cell administration, the EDSS score may remain the same or may decrease (e.g., by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0).

[0098] The various methods disclosed herein can be methods for improving walking, vision, balance, cognition or other symptoms in a subject (such as a subject having multiple sclerosis), and / or methods for improving the multiple sclerosis functional composite (MSFC), EDSS, or MSSS score in a subject (such as a subject having multiple sclerosis). Thus, in certain embodiments, the treatment methods disclosed herein include methods for stabilizing or improving a disorder or symptom (e.g., motor ability / balance / hand dexterity, sleep, vision or color vision, fatigue, urinary urgency) in a patient, such that the patient's disability score (when measured by these tests or another suitable test) at 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 3 months, 6 months, 1 year, or 2 years after treatment is at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 25%, at least about 40%, at least about 50% or even at least about 60% higher compared to the EDSS score before T cell therapy.

[0099] For example, the EDSS score of a subject can be tested by evaluating the subject's ability in tests at various time points (e.g., 0 months (baseline), 1 month, 2 months, 3 months, 6 months, 1 year and 2 years). In certain embodiments, if there is a recorded decrease in the subject's score after administration of T cells, then subsequently MS is considered stable and / or not progressing. In other embodiments, if there is no increase or decrease in the subject's score after administration of T cells, then subsequently MS is considered stable and / or not progressing. The EDSS test can be repeated at any time point from the start of CTL treatment (e.g., at 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months from the start of CTL treatment) to evaluate whether the treatment has slowed or stopped further deterioration of motor ability / balance / hand dexterity, sleep improvement, vision or color vision improvement, fatigue, urinary urgency.

[0100] In some embodiments, the progression of gait impairment can be examined by using a gait test to evaluate, for example, the ability of a subject in a 25-foot walk test at different time points. In certain embodiments, if there is no recorded worsening in gait, then subsequently the subject is considered to have no progressive worsening in gait. For patients who have not already received T cell therapy, subjects showing progressive gait impairment initiate treatment with T cells (e.g., CTLs). The gait test is repeated (e.g., at 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months from the start of treatment), and it can be evaluated whether the treatment decelerated or halted further worsening of gait ability (e.g., as measured by a gait test).

[0101] Whether there has been an improvement in cognitive outcomes associated with MS treatment, i.e., a deceleration of cognitive decline, stabilization of cognitive decline, or improvement in cognitive function, can be evaluated using the PASAT (e.g., PASAT 2 or PASAT 3) or SDMT test, or using the MS-COG test (see Erlanger et al., J Neuro Sci 340: 123-129 (2014)).

[0102] The actual dosage level of the active ingredient in the pharmaceutical compositions provided herein can be varied so as to achieve an amount of the active ingredient, composition, and method of administration that is effective in achieving the desired therapeutic response for a particular patient without being toxic to the patient.

[0103] The selected dosage level depends on various factors such as the activity of the particular agent used, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound used, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0104] [Example 1] Study of multiple treatments (therapies) for MS patients and EBV-specific T cell therapy for MS Participants provided 200 - 400 mL of blood samples. Peripheral blood mononuclear cells from this sample were used for the laboratory generation of autologous latent membrane protein (LMP1&2) / Epstein - Barr virus nuclear antigen 1 (EBNA1) - specific T cells, which were suspended in clinically - graded saline. The investigational drug was produced by stimulating gamma - irradiated autologous peripheral blood mononuclear cells infected with recombinant adenovirus vector AdE1 - LMPpoly, an adenovirus vector encoding multiple CD8+ T - cell epitopes from Epstein - Barr virus nuclear antigen - 1 (EBNA1), latent membrane protein 1 (LMP1), and LMP2A. Next, the T - cell cultures were evaluated for cell yield, viability, and T - cell frequency. Typically, it takes about 5 weeks from the collection of 200 - 400 mL of blood sample to the first cell administration.

[0105] Each patient received autologous T cells stimulated ex vivo to increase reactivity against EBNA1, LMP1, and LMP2A and was followed for 26 weeks. Patients received T - cell therapy intravenously at 2 - week intervals. Each dose was given once, with the first dose being 5×10 6 T cells, followed by doses of 1×10 7 T cells, 1.5×10 7 T cells, and 2×10 7 T cells. A total of 4 doses were given over 8 weeks. Each dose of cells was administered by intravenous line infusion, allowing for a slow infusion of T cells into the blood rather than a bolus of cells.

[0106] Thirteen patients were enrolled. Three patients were withdrawn before receiving cell therapy: one due to an unrelated diagnosis of malignancy and two due to the inability to generate EBV - specific T cells. The remaining 10 patients each received 4 T - cell infusions per protocol (Table 1 below).

[0107] Autologous EBV-CTLs were well tolerated, and no serious adverse events (AEs) were observed. Only one patient experienced an associated or probably associated AE, which was a transient grade 1 "taste change" judged to be due to the DMSO vehicle. Grade 4 or 5 AEs were not reported.

[0108] Six patients experienced symptomatic and objective clinical improvement, which began 2 - 14 weeks after the first infusion (Table 1). Reduction in fatigue was a prominent feature in patients with clinical improvement. A correlation between EBV reactivity and clinical response was observed. Patients who received T cells with high EBV reactivity experienced a greater clinical response. Six patients in this study received T cells with EBV reactivity of 7% or more. Five of these patients experienced clinical improvement, and three patients experienced improvement in the EDSS score. Four patients received T cells with EBV reactivity of 3% or less. Only one of these showed clinical improvement, one showed deterioration of the EDSS, and two reported no change. Clinical improvement or its absence during the 6 - month study period also correlated with the biomarker of EBV - specific T - cell function of the administered T cells (Figure 2). Patients who experienced clinical benefit received treatment in which EBV - specific CD8+ T cells expressing CD107a, IFNγ, and TNFα were significantly enriched compared to patients in whom no benefit was observed. Furthermore, clinical benefit correlated with the multifunctionality of the administered T cells (expression of CD107a, IFNγ, TNFα, and IL - 2).

[0109] Autologous T - cell therapy for MS patients in this study was safe, well tolerated, with no severe AEs, and treatment - related AEs were only dysgeusia, probably related to DMSO in the formulation rather than the T cells.

[0110] Clinical improvement in SPMS and PPMS patients was from a relatively stable baseline (up to 5 years) and did not represent resolution of acute MS inflammation. Of the six patients who received T cells with EBV reactivity of 7% or more, five showed clinical improvement and three patients improved their EDSS score.

[0111] Consistent with the hypothesized mechanism of action of EBV-specific T cells, there appears to be a dose-response correlation between the EBV reactivity of the T cell product and clinical improvement.

[0112] Reduction of fatigue was a consistent and prominent feature of responders. Fatigue is the most disabling symptom of MS and, in many cases, can precede clinical MS onset by several years.

[0113] The inventors' data are added to the extensive evidence for the pathogenic role of EBV infection in MS. Since T cells have access to all CNS compartments, T cell therapies that target only EBV-infected B cells are a new therapeutic approach that can provide favorable safety and durable efficacy.

[0114] [Example 2] Exemplary methods for multiparameter intracellular cytokine staining and degranulation analysis of LMP- and EBNA1-specific T cells 1. Dilute the test LMP / EBNA1 CD8 pepmix (100 μg / ml stock), EBNA1 pepmix (100 μg / ml stock), and any HLA-compatible peptide epitope (200 μg / mL stock) to 2 μg / mL with RPMI1640 - 10% FCS (the final concentration in the assay will be 1 μg / mL). 2. Dilute the cell stimulation cocktail 1:50 with RPMI1640 - 10% FCS (this will be a 1:100 final dilution in the assay). Note: The eBioscience cell stimulation cocktail is diluted 1:5 with RPMI, the stock concentration is multiplied by 100, and it is stored at -20 °C. 3. Add 100 μL of the appropriate pepmix, peptide epitope, or cell stimulation cocktail, or 100 μL of RPMI1640 - 10% FCS (no peptide control) to the appropriate wells of a 96-well V-bottom plate. 4. Dilute PBMC or T cells to 5 × 10 6 cells / mL with RPMI1640 - 10% FCS (this will result in 5 × 10 5 cells per assay). 5. Add GolgiPlug (brefeldin A) to the cells to bring the final ratio of the cells to 2 μL / mL (the final concentration of GolgiPlug in the assay is 1 μL / mL). 6. Add GolgiStop (monensin) to the cells to bring the final ratio of the cells to 1.4 μL / mL (the final concentration of GolgiStop in the assay is 0.7 μL / mL). 7. Add FITC-conjugated anti-CD107a to the cells to bring the final ratio to 50 μL / mL (the final amount of anti-CD107a is 5 μL / test). 8. Add 100 μL of the cell suspension per well to the required wells of a 96-well V-bottom plate. 9. Incubate at 37 °C / 6.5% CO2 for 4 hours. 10. Centrifuge the plate at 2300 rpm for 2 minutes. 11. Discard the supernatant. Add 200 μL of PBS-2% FCS per well to wash the cells and centrifuge the plate at 2300 rpm for 2 minutes. Repeat this step. 12. Wash the cells twice with 200 μL of PBS-2% FCS per well and centrifuge the tube at 1000 g (2300 rpm) for 2 minutes.

[0115] Surface cell antigen staining 13. Resuspend the cells in 50 μL / well of PBS-2% FCS containing 0.125 μL of perCP-Cy5.5-conjugated anti-CD8, 0.25 μL of Pacific Blue-conjugated anti-CD4, and 0.2 μL of Live / Dead Near IR. Incubate at 4 °C for 30 minutes. 14. Centrifuge the plate at 2300 rpm for 2 minutes. Discard the supernatant. Add 200 μL of PBS-2% FCS per well to wash the cells. Centrifuge the plate at 2300 rpm for 2 minutes. Repeat this step. 15. Resuspend the cells in 100 μL / well of BD cytofix / cytoperm solution and incubate at 4 °C for 20 minutes. 16. Centrifuge the plate at 2300 rpm for 2 minutes. Discard the supernatant. Add 200 μL of BD Perm / Wash per well to wash the cells. Centrifuge the plate at 2300 rpm for 2 minutes. Repeat this step.

[0116] Intracellular cytokine staining 17. Resuspend the fixed / permeabilized cells in 50 μL / well of BD Perm / Wash solution containing 1 μL of PE-conjugated anti-IL-2, 1 μL of AF700-conjugated anti-IFNγ, and 0.25 μL of APC-conjugated anti-TNF. Incubate at 4°C for 30 minutes. 18. Centrifuge the plate at 2300 rpm for 2 minutes. Discard the supernatant. Add 200 μL of Perm / Wash per well to wash the cells. Centrifuge the plate at 2300 rpm for 2 minutes. Repeat this step. 19. Resuspend the cells in 200 μL of PBS-2% paraformaldehyde and store at 4°C. 20. Acquire the cells using a BD Fortessa. Analyze cytokine production / degranulation using Flow Jo software.

[0117]

Table 2

Claims

1. A method for treating or preventing multiple sclerosis (MS) in a subject, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC.

2. The method of claim 1, wherein at least 5% of the CTLs express CD107a.

3. The method of claim 1, wherein at least 10% of the CTLs express CD107a.

4. The method of claim 1, wherein at least 15% of the CTLs express CD107a.

5. The method of claim 1, wherein at least 20% of the CTLs express CD107a.

6. The method of any one of claims 1 to 5, wherein at least 5% of the CTLs express IFNγ.

7. The method of claim 6, wherein at least 10% of the CTLs express IFNγ.

8. The method of claim 7, wherein at least 15% of the CTLs express IFNγ.

9. The method of claim 8, wherein at least 20% of the CTLs express IFNγ.

10. The method of any one of claims 1 to 9, wherein at least 5% of the CTLs express TNF.

11. The method of claim 10, wherein at least 10% of the CTLs express TNF.

12. The method of claim 11, wherein at least 15% of the CTLs express TNF.

13. The method of claim 12, wherein at least 20% of the CTLs express TNF.

14. The method of any one of claims 1 to 13, wherein at least 1% of the CTLs express IL-2.

15. The method of claim 14, wherein at least 5% of the CTLs express IL-2.

16. The method of claim 15, wherein at least 10% of the CTLs express IL-2.

17. The method of claim 16, wherein at least 15% of the CTLs express IL-2.

18. The method of claim 1, wherein at least 30% of the CTLs express CD107a, IFNγ, TNFα, and IL-2.

19. The method of claim 1, wherein at least 40% of the CTLs express CD107a, IFNγ, TNFα, and IL-2.

20. The method of claim 1, wherein at least 50% of the CTLs express CD107a, IFNγ, TNFα, and IL-2.

21. The method of claim 1, wherein at least 70% of the CTLs express CD107a, IFNγ, TNFα, and IL-2.

22. The method of any one of claims 1 to 21, wherein the CTLs have an EBV reactivity of at least 5%.

23. The method of claim 22, wherein the CTLs have an EBV reactivity of at least 7%.

24. The method of claim 23, wherein the CTLs have an EBV reactivity of at least 10%.

25. The method of claim 24, wherein the CTLs have an EBV reactivity of at least 15%.

26. The method of claim 25, wherein the CTLs have an EBV reactivity of at least 20%.

27. The method of claim 26, wherein the CTLs have an EBV reactivity of at least 30%.

28. 1. A method of treating or preventing multiple sclerosis (MS) in a subject, comprising: (a) isolating from a subject a sample containing cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC; (b) administering the CTL to a subject; The method includes:

29. 1. A method of treating or preventing MS in a subject, comprising: (a) incubating a sample containing autologous cytotoxic T cells (CTLs) with antigen-presenting cells (APCs) that present EBV peptides, thereby inducing proliferation of peptide-specific T cells in the sample; (b) administering peptide-specific autologous CTL to the subject; The method includes:

30. 1. A method of treating or preventing MS in a subject, comprising: (a) incubating antigen-presenting cells (APCs) with a nucleic acid construct encoding an EBV peptide, thereby inducing the APCs to present the EBV peptide; (b) inducing peptide-specific CTL proliferation by incubating the sample containing the autologous CTL with antigen-presenting cells (APCs), thereby inducing the autologous CTL to proliferate; and (c) administering peptide-specific autologous CTL to the subject. The method includes:

31. 31. The method of claim 30, wherein the nucleic acid construct is a viral vector.

32. The method of claim 31 , wherein the viral vector is AdE1-LMPpoly.

33. The method of any one of claims 29 to 32, further comprising analyzing the expression of CD107a by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to a subject if at least 5% of the expanded peptide-specific autologous CTLs express CD107a.

34. The method of claim 33, wherein the CTLs are administered if at least 10% of the expanded peptide-specific autologous CTLs in the sample express CD107a.

35. The method of claim 33 or 34, wherein if at least 15% of the expanded peptide-specific autologous CTLs in the sample express CD107a, the CTLs are administered.

36. The method of any one of claims 33 to 35, wherein if at least 20% of the expanded peptide-specific autologous CTLs in the sample express CD107A, the CTLs are administered.

37. The method according to any one of claims 29 to 36, further comprising analyzing the expression of IFNγ by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to a subject if at least 5% of the expanded peptide-specific autologous CTLs express IFNγg.

38. 38. The method of claim 37, wherein if at least 10% of the expanded peptide-specific autologous CTLs in the sample express IFNγ, the CTLs are administered.

39. The method of claim 37 or 38, wherein if at least 15% of the expanded peptide-specific autologous CTLs in the sample express IFNγ, the CTLs are administered.

40. 40. The method of any one of claims 37 to 39, wherein if at least 20% of the expanded peptide-specific autologous CTLs in the sample express IFNγ, the CTLs are administered.

41. The method according to any one of claims 29 to 40, further comprising analyzing the expression of TNF by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to a subject if at least 5% of the expanded peptide-specific autologous CTLs express TNF.

42. The method of claim 41, wherein if at least 10% of the expanded peptide-specific autologous CTLs in the sample express TNF, the CTLs are administered.

43. The method of claim 41 or 42, wherein if at least 15% of the expanded peptide-specific autologous CTLs in the sample express TNF, the CTLs are administered.

44. 44. The method of any one of claims 41 to 43, wherein if at least 20% of the expanded peptide-specific autologous CTLs in the sample express TNF, the CTLs are administered.

45. The method according to any one of claims 29 to 44, further comprising analyzing the expression of IL-2 by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to a subject if at least 1% of the expanded peptide-specific autologous CTLs express IL-2.

46. The method of claim 45, wherein if at least 5% of the peptide-specific autologous CTLs in the sample express IL-2, the CTLs are administered.

47. 47. The method of claim 45 or 46, wherein if at least 10% of the peptide-specific autologous CTLs in the sample express IL-2, the CTLs are administered.

48. 48. The method of any one of claims 45 to 47, wherein if at least 15% of the expanded peptide-specific autologous CTLs in the sample express IL-2, the CTLs are administered.

49. The method of any one of claims 29 to 32, further comprising analyzing the expression of CD107a, TNF, IFNγ, and IL-2 by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to a subject if at least 20% of the expanded peptide-specific autologous CTLs express CD107a, TNF, IFNγ, and IL-2.

50. 50. The method of claim 49, wherein if at least 30% of the expanded peptide-specific autologous CTLs in the sample express CD107a, TNF, IFNγ, and IL-2, the CTLs are administered.

51. The method of claim 49 or 50, wherein if at least 40% of the expanded peptide-specific autologous CTLs in the sample express CD107a, TNF, IFNγ, and IL-2, the CTLs are administered.

52. 52. The method of any one of claims 49 to 51, wherein if at least 50% of the expanded peptide-specific autologous CTLs in the sample express CD107a, TNF, IFNγ, and IL-2, the CTLs are administered.

53. The method according to any one of claims 29 to 52, further comprising analyzing the EBV reactivity of the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to a subject if the reactivity is above a predetermined threshold.

54. 54. The method of claim 53, wherein the threshold is 5%.

55. 54. The method of claim 53, wherein the threshold is 7%.

56. 54. The method of claim 53, wherein the threshold is 10%.

57. 54. The method of claim 53, wherein the threshold is 20%.

58. 54. The method of claim 53, wherein the threshold is 30%.

59. 54. The method of claim 53, wherein the threshold is 50%.

60. 60. The method of any one of claims 29 to 59, wherein the sample is incubated with one or more cytokines in step (a).

61. 61. The method of any one of claims 29 to 60, wherein the APC comprises a B cell.

62. 62. The method of any one of claims 29 to 61, wherein the APC comprises an antigen-presenting T cell.

63. 63. The method of any one of claims 29 to 62, wherein the APC comprises a dendritic cell.

64. 64. The method of any one of claims 29 to 63, wherein the APC comprises an artificial antigen presenting cell.

65. The method of claim 64, wherein the artificial antigen presenting cell is an aK562 cell.

66. 66. The method of any one of claims 28 to 65, wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

67. 67. The method of any one of claims 1 to 66, further comprising obtaining a sample from a subject.

68. 68. The method of any one of claims 1 to 67, wherein the EBV peptide comprises an amino acid sequence listed in Table 1.

69. 68. The method of any one of claims 1 to 67, wherein the EBV peptide comprises an LMP1 peptide or a fragment thereof.

70. 68. The method of any one of claims 1 to 67, wherein the EBV peptide comprises an LMP2A peptide or a fragment thereof.

71. 68. The method of any one of claims 1 to 67, wherein the EBV peptide comprises an EBNA1 peptide or a fragment thereof.

72. Approximately 5 × 10 per dose 6 72. The method of any one of claims 1 to 71, comprising administering to a subject the CTL.

73. Approximately 1 × 10 per dose 7 72. The method of any one of claims 1 to 71, comprising administering to a subject the CTL.

74. Approximately 1.5 × 10 per dose 7 72. The method of any one of claims 1 to 71, comprising administering to a subject the CTL.

75. Approximately 2 × 10 per dose 7 72. The method of any one of claims 1 to 71, comprising administering to a subject the CTL.

76. 76. The method of any one of claims 1 to 75, wherein multiple doses of the CTLs are administered to the subject, for example in dose escalation.

77. 77. The method of claim 76, wherein the dose is administered weekly.

78. 77. The method of claim 76, wherein the dose is administered every other week.

79. 80. The method of claim 78, comprising administering four successively greater doses of CTLs.

80. 5×10 6 First dose of CTLs: 1 x 10 7 The second dose of CTLs, 1.5 x 10 7 and the third dose of 2 x 10 CTLs. 7 80. The method of claim 79, comprising administering a fourth dose of the CTLs.

81. 81. The method of any one of claims 1 to 80, wherein the subject does not experience significant adverse effects as a result of CTL administration.

82. further comprising evaluating the efficacy of adoptive immunotherapy in a subject with multiple sclerosis; (a) obtaining a first sample of cerebrospinal fluid (CSF) from a subject; (b) determining the amount of anti-EBV IgG in the CSF in the first sample before administration of the CTL; (c) obtaining a second sample of CSF from the subject after a period of time following administration of the CTLs; (d) determining the amount of anti-EBV IgG in the CSF in the second sample; Including, The method, wherein if the amount of anti-EBV IgG in the second sample is less than in the first sample, the disease is stable and / or not progressing.

83. 83. The method of any one of claims 1 to 82, wherein the MS is relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS.

84. 84. The method of claim 83, wherein the MS is primary progressive MS.

85. 85. The method of any one of claims 1 to 84, wherein the subject's motor skills / balance / dexterity, sleep, eyesight or color vision, fatigue, and / or urinary urgency are improved following CTL administration.

86. 86. The method of any one of claims 1 to 85, wherein the subject's EDSS score remains the same after CTL administration.

87. 86. The method of any one of claims 1 to 85, wherein the subject's EDSS score is reduced by at least 0.5 following CTL administration.

88. 86. The method of any one of claims 1 to 85, wherein the subject's EDSS score is reduced by at least 1.0 following CTL administration.

89. A method for reducing anti-EBV IgG levels in the CSF in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC.

90. A method for improving vision, improving color vision, or stabilizing vision loss in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC.

91. A method for improving motor skills, balance, or manual dexterity in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC.

92. A method for improving sleep in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC.

93. A method for treating or preventing fatigue in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC.

94. A method for treating or preventing urinary urgency in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC.

95. 95. The method of any one of claims 1 to 94, wherein MS is relapsing-remitting MS.

96. 95. The method of any one of claims 1 to 94, wherein MS is secondary progressive MS.

97. 95. The method of any one of claims 1 to 94, wherein MS is primary progressive MS.

98. 95. The method of any one of claims 1 to 94, wherein the MS is primary progressive progressive relapsing MS.

99. 1. A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a sample comprising T cells from a subject; (b) isolating autologous T cells in the sample; (c) determining EBV reactivity of autologous T cells in the sample and selecting the subject for adoptive immunotherapy if at least a threshold percentage of the autologous T cells are EBV reactive. The method includes:

100. 100. The method of claim 99, wherein the threshold is 1%.

101. 100. The method of claim 99, wherein the threshold is 2%.

102. 100. The method of claim 99, wherein the threshold is 3%.

103. 100. The method of claim 99, wherein the threshold is 5%.

104. 100. The method of claim 99, wherein the threshold is 10%.

105. 100. The method of claim 99, wherein the threshold is 20%.

106. 1. A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a sample comprising T cells from a subject; (b) isolating autologous T cells in the sample; (c) determining the percentage of autologous T cells in the sample that express CD107A, and selecting the subject for adoptive immunotherapy if at least a particular percentage of the autologous T cells express CD107A. The method includes:

107. The method of claim 106, wherein the subject is selected for adoptive immunotherapy if at least 1% of autologous T cells express CD107A.

108. The method of claim 106, wherein the subject is selected for adoptive immunotherapy if at least 3% of autologous T cells express CD107A.

109. The method of claim 106, wherein the subject is selected for adoptive immunotherapy if at least 5% of autologous T cells express CD107A.

110. The method of claim 106, wherein the subject is selected for adoptive immunotherapy if at least 10% of autologous T cells express CD107A.

111. 1. A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a sample comprising T cells from a subject; (b) isolating autologous T cells in the sample; (c) determining the percentage of autologous T cells in the sample that express TNF and selecting the subject for adoptive immunotherapy if at least a particular percentage of the autologous T cells express TNF. The method includes:

112. The method of claim 111, wherein the subject is selected for adoptive immunotherapy if at least 1% of autologous T cells express TNF.

113. The method of claim 111, wherein the subject is selected for adoptive immunotherapy if at least 3% of autologous T cells express TNF.

114. The method of claim 111, wherein the subject is selected for adoptive immunotherapy if at least 5% of autologous T cells express TNF.

115. The method of claim 111, wherein the subject is selected for adoptive immunotherapy if at least 10% of autologous T cells express TNF.

116. 1. A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a sample comprising T cells from a subject; (b) isolating autologous T cells in the sample; (c) determining a percentage of autologous T cells in the sample that express IFNγ, and selecting the subject for adoptive immunotherapy if at least a particular percentage of the autologous T cells express IFNγ. The method includes:

117. The method of claim 116, wherein the subject is selected for adoptive immunotherapy if at least 1% of autologous T cells express IFNγ.

118. The method of claim 116, wherein the subject is selected for adoptive immunotherapy if at least 3% of autologous T cells express IFNγ.

119. The method of claim 116, wherein the subject is selected for adoptive immunotherapy if at least 5% of autologous T cells express IFNγ.

120. The method of claim 116, wherein the subject is selected for adoptive immunotherapy if at least 10% of autologous T cells express IFNγ.

121. 1. A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a sample comprising T cells from a subject; (b) isolating autologous T cells in the sample; (c) determining a percentage of autologous T cells in the sample that express IL-2, and selecting the subject for adoptive immunotherapy if at least a particular percentage of the autologous T cells express IL-2. The method includes:

122. The method of claim 116, wherein the subject is selected for adoptive immunotherapy if at least 1% of autologous T cells express IL-2.

123. The method of claim 116, wherein the subject is selected for adoptive immunotherapy if at least 3% of autologous T cells express IL-2.

124. The method of claim 116, wherein the subject is selected for adoptive immunotherapy if at least 5% of autologous T cells express IL-2.

125. The method of claim 116, wherein the subject is selected for adoptive immunotherapy if at least 10% of autologous T cells express IL-2.

126. 1. A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a PMBC sample from a subject; (b) isolating autologous T cells in the sample; (c) determining a percentage of autologous T cells in the sample that express CD107a, IFNγ, TNFα, and IL-2, and selecting the subject for adoptive immunotherapy if at least a certain percentage of the autologous T cells express CD107a, IFNγ, TNFα, and IL-2. The method includes:

127. The method of claim 126, wherein the subject is selected for adoptive immunotherapy if at least 1% of autologous T cells express CD107a, IFNγ, TNFα, and IL-2.

128. The method of claim 126, wherein the subject is selected for adoptive immunotherapy if at least 3% of autologous T cells express CD107a, IFNγ, TNFα, and IL-2.

129. The method of claim 126, wherein the subject is selected for adoptive immunotherapy if at least 5% of autologous T cells express CD107a, IFNγ, TNFα, and IL-2.

130. The method of claim 126, wherein the subject is selected for adoptive immunotherapy if at least 10% of autologous T cells express CD107a, IFNγ, TNFα, and IL-2.

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