Methods for stratifying and treating multiple sclerosis

Abstract

The present disclosure relates to the field of multiple sclerosis (MS) stratification by analyzing body fluids of MS patients for CD27-Th1 CD4+ cells in the body fluids, such as blood or CSF. The present invention also relates to the field of antigen-specific immunotherapy for MS, such as induction of tolerance involving GDP-L-fucose synthase for responders. The objective of the present invention is to improve stratification of MS patients in order to develop individualized treatment regimens. The objective of the present invention is also to develop antigen-specific tolerization strategies, particularly for treating certain MS patient subpopulations.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present disclosure relates to the field of multiple sclerosis (MS) stratification by analysis of body fluids of MS patients. The present invention also relates to the field of antigen-specific immunotherapy, including induction of tolerance. [Background technology]

[0002] 2. Background of the Invention Multiple sclerosis (MS) is a destructive autoimmune inflammatory disease that primarily affects young adults. MS is a classic example of an organ-specific autoimmune disease (AID) because the autoimmune response targets only the central nervous system (CNS), which consists of the brain and spinal cord. Organ-specific AID means that the patient's immune system damages specific tissue or cell types with autoreactive T cells and / or antibodies.

[0003] MS preferentially affects young adults between the ages of 20 and 40, but children and the elderly can also develop MS. The disease is approximately two to three times more common in women than in men. MS usually becomes clinically manifested by temporary problems with vision (acute optic neuritis), sensory, or motor and autonomic function, but can lead to a wide range of neurological symptoms.

[0004] At the time of first manifestation, if differential diagnoses have been excluded, the disease is termed clinically initial stage (CIS), provided that cerebrospinal fluid (CSF) and magnetic resonance imaging (MRI) findings are consistent with the diagnosis. MRI reveals lesions in locations typical of MS (i.e., juxtacortical, periventricular, brainstem or spinal cord). Relapsing-remitting multiple sclerosis (RRMS) can be diagnosed if certain criteria are met, which can be summarized as spatially multifocal (more than one lesion or clinical symptom / sign) and temporally multifocal (more than one event). The spatial scenario is the chance discovery of MRI lesions compatible with MS without clinical symptoms. This is termed radiologically isolated syndrome (RIS), which can be considered a precursor to CIS and RRMS. More than 80% of patients will suffer from one of these, with the majority of patients subsequently developing the so-called secondary progressive MS (SPMS). At this point, relapses / exacerbations become less frequent or stop completely, and neurological disability gradually increases with or without relapses.

[0005] A special type of MS is primary progressive MS (PPMS), which does not experience relapses, but rather begins with a steady deterioration of neurological symptoms (e.g., walking ability). PPMS affects approximately 10% of MS patients, with equal frequency in men and women. Its onset is usually later than CIS or RRMS. With regard to causes and disease pathogenesis, PPMS is considered similar to the RIS-CIS-RRMS-SPMS described above.

[0006] Typically, MS is diagnosed according to the revised McDonald criteria or more recently the Lublin criteria, which are able to distinguish different forms and disease activity of MS (Thompson et al., 2018, Lancet Neurol, 17(2):162-173).

[0007] MS is a disease with a complex genetic background. Over 200 MS risk alleles or quantitative traits (common variants in genes detected as single nucleotide polymorphisms (SNPs)) have been identified in the last decade, however, by far the most important is the human leukocyte antigen (HLA)-DR15 haplotype. In addition, several environmental / lifestyle risk factors have been found. These include, most importantly, Epstein-Barr virus (EBV) infection, smoking, low vitamin D3 levels, and obesity.

[0008] All genetic and environmental risk factors are common and shared by many individuals in the healthy population. The exact reason why individuals with certain genetic and environmental risk factors develop the disease is unclear, but it is thought that viral and bacterial infections due to changes in the gut microbiome may be a trigger, for example. Compared to a risk of 1 / 1000 in the general population, the concordance rate in identical twins is 10-30%, and the risk for first-degree relatives of MS patients is approximately 2-4%, giving an estimate of genetic risk versus environmental risk, but the interaction between these two factors is also complex.

[0009] To identify the components of the CNS where the autoimmune response in MS is directed, researchers have focused on cells and structures affected by MS (particularly myelin and axons / neurons) and on proteins specific to these cells / structures. Over the last 30 years, several myelin proteins (such as myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendroglial glycoprotein (MOG)) have been identified as encephalitogenic in animal models (experimental autoimmune encephalomyelitis; EAE) (i.e., injection into susceptible rodent strains leads to a disease similar to MS) and also by examination of immune cells from MS patients (Sospedra and Martin, 2005, Annu Rev Immunol, 23:683-747). The above autoantigens are expressed in the brain specifically and exclusively (PLP and MOG) or almost exclusively (MBP) in the CNS. In MS, a few autoantigens that are not CNS specific (such as alpha-B crystallin and transaldolase-H) have also been described as potential targets.

[0010] Recently, a further MS-associated antigen has been identified (GDP-L-fucose synthase (GDP-L-FS); WO2020 / 002674). This protein has been found to be immunodominant in MS and is an autoantigen.

[0011] Further evidence implicates CD4+ autoreactive T cells as central factors in the autoimmune pathogenesis of MS, possibly involved not only in the induction and maintenance of the autoimmune response but also during tissue damage (Sospedra and Martin, 2005). The frequency of high avidity CD4+ T cell responses against the main components of the myelin sheath (such as MBP, PLP, and MOG) is increased in MS patients (Bielekova et al., 2004, J Immunol, 172:3893-3904). Due to their role in disease pathogenesis, CD4+ T cells are a target for therapeutic intervention.

[0012] Close investigation of the immune response to CNS-specific proteins has shown that certain peptides are recognized by the majority of patients in the context of disease-associated HLA-DR molecules: such peptides are termed immunodominant (Bielekova et al., 2004).

[0013] The following characteristics indicate that certain peptides of a protein are immunodominant from an MS perspective: a) frequent recognition of this peptide by T cells (i.e., by approximately 10% or more of MS patients), often in the context of disease-associated HLA alleles or haplotypes (Sospedra and Martin, 2005), and b) Recognition of this peptide by disease-associated T cells, such as those that respond to the peptide at low concentrations (high avidity T cells) (Bielekova et al., 2004) and are therefore considered particularly dangerous and / or that have a pro-inflammatory phenotype and / or that are isolated from the target organ or compartment (CNS) (in the case of MS, T cells infiltrating the brain, spinal cord or CSF).

[0014] However, high avidity recognition is not a prerequisite, as low avidity myelin-specific T cells have also been shown to be pathogenic in humanized transgenic mouse models (Quandt et al. 2012, J Immunol, 189(6):2897-2908).

[0015] It has recently been demonstrated that T cells from MS patients undergo increased in vitro expansion in the absence of exogenous antigens (Mohme et al., 2013, Brain, 136:1783-1798). These "autologous" T cells are enriched in cells present in the CNS compartment of MS patients and can therefore be considered a peripheral blood source of brain / CSF-infiltrating T cells (Jelcic et al., 2018, Cell, 175(1):85-100.e23).

[0016] When data from in vitro T cell tests are not available or in addition to such tests, the immune recognition of peptides can also be predicted / predicted from peptides that are considered to bind well to the HLA-class I or class II alleles of an individual, and for CD8+ T cells and CD4+ T cells, respectively.Peptide binding prediction is well known to those skilled in the art.Peptide binding can be predicted by established prediction algorithms (NetMHCII-www.cbs.dtu.dk / services / NetMHCII / ; IEDB-www.iedb.org / ) and by analyzing HLA binding motifs (SYFPEITHI-www.syfpeithi.de / ).

[0017] Immunodominant peptides can be used in antigen-specific immunotherapy, such as tolerance induction.One example is European Patent No. 2205273, which discloses the immunodominant peptides of MBP, PLP and MOG and their application for MS treatment.In the approach disclosed herein, peptides are bound to white blood cells or red blood cells.

[0018] Tolerance induction is antigen-specific and either renders autoreactive T cells non-functional or anergic, or induces regulatory T (Treg) cells that specifically suppress inappropriate autoimmunity against the target antigen. Induction of tolerance to a target autoantigen is a very important therapeutic goal in autoimmune diseases. Tolerance induction provides an opportunity to specifically weaken pathogenic autoimmune responses in an effective manner with few side effects. Also, instead of or in addition to immunodominant peptides being fragments of proteins, tolerance induction can be obtained by applying whole proteins (Kennedy et al., 1990, J Immunol, 144(3):909-915).

[0019] Some pathological features of MS are reflected in EAE models, a line of animal models of Th1 / Th17 cell-driven autoimmune diseases. Studies of relapsing EAE (R-EAE) in SJL mice clearly show that chronic demyelination involves the activation of T cell responses against an immunodominant myelin peptide (i.e., PLP139-154) that dictates initial disease exacerbation. The immune response subsequently expands to other myelin peptides of PLP, MBP, and MOG, a process termed epitope spreading. Non-responsiveness (i.e., tolerance) to T cells can be induced, for example, when antigen-peptide-pulsed antigen-presenting cells (APCs) are treated with, for example, the crosslinker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI; also abbreviated as EDC).

[0020] In preclinical experiments, a single iv injection of naive mouse splenocytes pulsed with a mixture of encephalitogenic myelin peptides and fixed with the crosslinker EDC proved highly effective in inducing peptide-specific tolerance in vivo. In EAE, this protocol not only prevented disease in animals, but also effectively reduced the occurrence and severity of all subsequent relapses when administered after disease induction, indicating that specific tolerance can downregulate ongoing autoimmune responses (Miller et al., 1991, Acad Sci, 636:79-94). Studies in EAE, which are more relevant to the treatment of MS, have shown that a cocktail of encephalitogenic myelin peptides can be used to simultaneously induce tolerance to multiple epitopes, thus targeting autoreactive T cells with multiple specificities.

[0021] Tolerization of human T cells with EDC-treated autoantigen-bound cells (e.g., APCs) (Vandenbark et al., 2000, Int Immunol, 12:57-66) or non-nucleated cells (i.e., red blood cells (RBCs)) is effective in vitro, as demonstrated by the inability of tolerized T cells to proliferate or produce Th1 cytokines and reduced expression of costimulatory molecules on these cells.

[0022] There is evidence that at least two distinct mechanisms are involved in the induction of antigen-specific tolerance by this regimen: 1) direct tolerance, when Th1 clones encountering nominal antigen / MHC complexes on antigen-bound APCs are anergized or deleted as a result of their inability to receive appropriate CD28-mediated costimulation; and 2) indirect mechanisms such as cross-tolerance (where tolerance is induced by re-processing and re-presentation of antigens by tolerogenic host APCs and / or expansion of Treg cells).

[0023] The latter cross-tolerance may be involved in the induction and / or expansion of antigen-specific Treg cells, a hypothesis also supported by the data obtained in the Phase Ib study disclosed herein. Furthermore, treatment of cells with EDC induces apoptosis in a significant proportion of treated cells. Thus, there may be an indirect mechanism involving APCs taking up fixed cells undergoing apoptosis (which are then processed and presented by host APCs). This is further supported by the effective induction of tolerance in MHC-deficient allogeneic mice. In-vitro bone marrow-derived dendritic cells effectively phagocytose and process fixed APCs pulsed with antigen.

[0024] Currently approved MS treatments involve various antigen-nonspecific immunomodulatory or immunosuppressive strategies, which are only partially effective. All current treatments require daily oral administration or injections / infusions at various intervals for extended periods of time. Moreover, these treatments are associated with numerous and sometimes severe side effects.

[0025] Treatments addressing the pathogenesis of MS at its root should aim to specifically delete or inhibit the function of pathogenic autoreactive cells without altering the "normal" immune system. This is important because global immunomodulation and / or immunosuppression comes at the cost of inhibiting beneficial regulatory and immune cells that perform defensive functions against pathogens.

[0026] Ideally, treatment should be individualized taking into account the patient's specific characteristics, such as their genetic background or the ability of their immune system to respond to certain antigens.

[0027] Thereby, personalized therapy can help improve patient outcomes. For personalized treatment regimens, MS stratification (i.e., identification of subtypes that are particularly responsive to certain treatments) is important. [Prior art documents] [Patent documents]

[0028] [Patent Document 1] International Publication No. 2020 / 002674 [Patent Document 2] European Patent No. 2205273 [Non-patent literature]

[0029] [Non-Patent Document 1] Thompson et al.,2018,Lancet Neurol,17(2):162-173 [Non-Patent Document 2] Sospedra and Martin,2005,Annu Rev Immunol,23:683-747 [Non-Patent Document 3] Bielekova et al.,2004,J Immunol,172:3893-3904 [Non-Patent Document 4] Quandt et al.2012,J Immunol,189(6):2897-2908 [Non-Patent Document 5] Mohme et al.,2013,Brain,136:1783-1798 [Non-Patent Document 6] Jelcic et al.,2018,Cell,175(1):85-100.e23 [Non-Patent Document 7] NetMHCII - www.cbs.dtu.dk / services / NetMHCII / [Non-Patent Document 8] IEDB - www.iedb.org / [Non-Patent Document 9] SYFPEITHI - www.syfpeithi.de / [Non-Patent Document 10] Kennedy et al., 1990, J Immunol, 144(3):909-915 [Non-Patent Document 11] Miller et al.,1991,Acad Sci,636:79-94 [Non-Patent Document 12] Vandenbark et al.,2000,Int Immunol,12:57-66 Summary of the Invention

[0030] Summary of the Invention It is an object of the present invention to improve the stratification of MS patients in order to develop individualized treatment regimens, and to develop antigen-specific tolerization strategies, especially for treating certain MS patient subpopulations.

[0031] In a first aspect of the present invention, there is provided a method for stratifying multiple sclerosis (MS) patients, comprising the steps of: - obtaining a body fluid, in particular blood, preferably peripheral blood, or cerebrospinal fluid (CSF) from an MS patient; and - detecting CD27-Th1CD4+ cells in the body fluid A method is provided, comprising:

[0032] In certain embodiments, the method comprises: - detecting the responsiveness of T cells and / or antibodies in the body fluid to the protein GDP-L-fucose synthase (GDP-L-FS), or to fragments, derivatives and / or splice variants thereof. Further includes:

[0033] In a further particular embodiment, the protein GDP-L-FS is a) having the amino acid sequence set forth in SEQ ID NO:1; b) has an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1; c) having an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1; d) has an amino acid sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the amino acid sequence set forth in SEQ ID NO:1, and the protein, or a fragment or splice variant thereof, binds to autologous HLA alleles and is recognized by T cells and / or is recognized by antibodies that bind to or recognize the amino acid sequence set forth in SEQ ID NO:1 or a fragment thereof; or e) TSTA3 gene, in particular encoded by the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11 or encoded by a gene that is at least 80%, preferably at least 90%, even more preferably at least 95% identical to the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11.

[0034] In one embodiment, the HLA allele is HLA allele DRB3 * 02:02 or HLA allele DRB3 * It is 03:01.

[0035] Preferably, the fragment comprises 5 to 50, preferably 5 to 20, more preferably 10 to 15 amino acids, even more preferably 15 amino acids.

[0036] Preferably, the fragment is a) is at least 85%, preferably at least 90%, more preferably at least 95% identical to the respective corresponding amino acid sequence; b) are at least 70%, preferably at least 80%, more preferably at least 90% homologous to their respective corresponding amino acid sequences; or c) are at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to their respective corresponding amino acid sequences, bind to autologous HLA alleles, are recognized by T cells, and / or are recognized by antibodies that bind to or recognize said respective amino acid sequences.

[0037] In a further preferred embodiment, the fragment comprises a sequence selected from the group comprising SEQ ID NO: 2 to 6 and SEQ ID NO: 37, preferably consisting of a sequence selected from the group comprising SEQ ID NO: 2 to 6 and SEQ ID NO: 37. In another preferred embodiment, the fragment comprises or consists of any sequence within the sequence defined by SEQ ID NO: 37.

[0038] In a second aspect of the present invention, there is provided a GDP-L-FS protein, or a fragment, derivative or splice variant thereof, as defined above, or a nucleotide sequence encoding a GDP-L-FS protein, or a fragment, derivative or splice variant thereof, for use in the treatment of MS in MS patients, in which CD27-Th1CD4+ cells are detected in a body fluid previously obtained from the MS patient, in particular blood, preferably peripheral blood, or CSF.

[0039] In a particular embodiment, T cells and / or antibodies previously obtained from body fluids of MS patients respond to the protein GDP-L-FS, or fragments, derivatives and / or splice variants thereof.

[0040] In a third aspect of the present invention, there is provided at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence as defined above for use in a method for inducing antigen-specific tolerance to autoantigens in MS patients, in which CD27-Th1CD4+ cells are detected in a body fluid previously obtained from the MS patient, in particular blood, preferably peripheral blood or CSF, and / or there is provided at least one carrier bound to at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence as defined above.

[0041] In a particular embodiment, T cells and / or antibodies previously obtained from body fluids of MS patients respond to the protein GDP-L-FS, or fragments, derivatives and / or splice variants thereof.

[0042] In a further particular embodiment, at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence and / or at least one carrier bound to at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence is applied by nasal, inhalation, oral, subcutaneous (sc), intracavity (ic), intramuscular (im), intradermal (id), transdermal (td) or intravenous (iv) administration, preferably by iv, sc, id, td, oral, inhalation or nasal administration.

[0043] In a fourth aspect of the present invention, there is provided CD27-Th1CD4+ cells for use in a method of monitoring a response to a method for inducing antigen-specific tolerance as disclosed above, wherein the CD27-Th1CD4+ cells are detected in a body fluid previously obtained from an MS patient, in particular blood, preferably peripheral blood, or CSF.

[0044] In a particular embodiment, it is furthermore intended to detect the reactivity of T cells and / or antibodies previously obtained from body fluids of MS patients against the protein GDP-L-FS as defined above, or against fragments, derivatives and / or splice variants thereof.

[0045] Preferably, the CD27-Th1CD4+ cells are additionally negative for the markers CCR7 and / or CD45RA.

[0046] In certain embodiments, the MS patient has the following characteristics: - Inflammation and / or neurodegeneration in the central nervous system specifically characterized by Gd-contrast enhanced T1 lesions and / or FLAIR T2 lesions, - higher expression of genes associated with Th1 cells or cytotoxicity and / or genes encoding proinflammatory cytokines such as IL-2 and / or IFN-γ compared to healthy controls, -HLA allotype HLA-DRB3 * 02:02 or DRB3 * 03:01 The present invention has one or more of the following:

[0047] In another aspect, a method for stratifying MS patients is described herein, comprising detecting CD27-Th1CD4+ cells in a sample obtained from the patient, thereby stratifying the patient.

[0048] In one embodiment, the sample comprises a bodily fluid.

[0049] In one embodiment, the bodily fluid comprises blood, eg, peripheral blood, or cerebrospinal fluid (CSF).

[0050] In one embodiment, the method comprises detecting the responsiveness of T cells and / or antibodies in said bodily fluid to the protein GDP-L-fucose synthase (GDP-L-FS), or a fragment, derivative, and / or splice variant thereof.

[0051] In another aspect, described herein is a method for treating an MS patient, comprising the steps of detecting CD27-Th1CD4+ cells in a sample obtained from the patient and administering an MS therapy to the patient, thereby treating the patient.

[0052] In one embodiment, the T cells and / or antibodies previously obtained from the patient's body fluid respond to the protein GDP-L-FS, or a fragment, derivative and / or splice variant thereof.

[0053] In one embodiment, the MS treatment comprises an immunodominant peptide.

[0054] In one embodiment, MS treatment involves treating a patient with antigen-specific immunotherapy (such as tolerance induction).

[0055] In one embodiment, treatment of a patient comprises administering to the patient an immunodominant peptide selected from MBP, PLP, and MOG, for example as disclosed in EP2205273.

[0056] In one embodiment, treatment of a patient comprises administering to the patient an immunodominant protein or peptide selected from GDP-L-FS, or a fragment, derivative, or splice variant thereof, e.g., as disclosed in WO2020 / 002674, and a protein from the RASGRP family, or a fragment, derivative, or splice variant thereof.

[0057] In one embodiment, the immunodominant peptide is chemically coupled to, for example, a white or red blood cell.

[0058] In one embodiment, the sample comprises a bodily fluid.

[0059] In one embodiment, the bodily fluid comprises blood, eg, peripheral blood, or cerebrospinal fluid (CSF). [Brief description of the drawings]

[0060] [Figure 1A]Figure 1. Flow cytometry gating strategy. (A-C) Doublets are first excluded, followed by identification of lymphocytes by size. A. CD3- are identified, and among these, plasma cells (CD19-CD138+), plasmablasts (CD19+CD138+), B cells (CD19+CD138-), and CD19-CD138- cells are identified. Among B cells, naive B cells (IgD+CD27-), unswitched memory B cells (IgD+CD27+), switched memory B cells (IgD-CD27+), and double negative (IgD-CD27-) B cell subsets are also identified. B. In CD3+ T cells, CD3+CD8+ cells are first identified and then separated into CM (CCR7+CD45RA-), EM (CCR7-CD45RA-), TEMRA (CCR7-CD45RA+), and naive (CCR7+CD45RA+). Then, CM, EM, and TEMRA CD8+ T cells are separated into CD28+ and CD28-. Each one of these CD8+ T cells is first separated into CCR6- and CCR6+, then into Th1 (CCR6-CCR4-CRTH2-), Th2-A (CCR6-CCR4+CRTH2-), Th2-B (CCR6-CCR4+CRTH2+), CCR6-CCR4-CRTH2+, Th1* (CCR6+CCR4-CRTH2-), Th17 (CCR6-CCR4+CRTH2-), CCR6+CCR4+CRTH2+, and CCR6+CCR4-CRTH2+ cells. C.CD3+ T cells, CD3+CD4+ cells are first identified, then into CM (CCR7+CD45RA-), EM (CCR7-CD45RA-), TEMRA (CCR7-CD45RA+), and naive (CCR7+CD45RA+). CM, EM, and TEMRA CD4+ T cells are then separated into CD28+CD27+, CD28+CD27-, and CD28-.Each one of these CD4+ T cells was first separated into CCR6- and CCR6+, then into Th1 (CCR6-CCR4-CRTH2-), Th2-A (CCR6-CCR4+CRTH2-), Th2-B (CCR6-CCR4+CRTH2+), CCR6-CCR4-CRTH2+, Th1* (CCR6+CCR4-CRTH2-), Th17 (CCR6-CCR4+CRTH2-), CCR6+CCR4+CRTH2+, and CCR6+CCR4-CRTH2+ cells. SPHERO™ AccuCount Particles were used to determine absolute counts. Antibodies: anti-CD3 AF700, anti-CD4 PE TR, anti-CD8 BV510, anti-CD45RA BV711, anti-CCR7 BV421, anti-CD27 APC Cy7, anti-CD28 PE Cy7, anti-CCR4 APC, anti-CRTh2 PE, anti-CCR6 BV785, anti-CD19 PerCPCy5.5, anti-IgD BV605, and anti-CD138 FITC. [Figure 1B]Figure 1. Flow cytometry gating strategy. (A-C) Doublets are first excluded, followed by identification of lymphocytes by size. A. CD3- are identified, and among these, plasma cells (CD19-CD138+), plasmablasts (CD19+CD138+), B cells (CD19+CD138-), and CD19-CD138- cells are identified. Among B cells, naive B cells (IgD+CD27-), unswitched memory B cells (IgD+CD27+), switched memory B cells (IgD-CD27+), and double negative (IgD-CD27-) B cell subsets are also identified. B. In CD3+ T cells, CD3+CD8+ cells are first identified and then separated into CM (CCR7+CD45RA-), EM (CCR7-CD45RA-), TEMRA (CCR7-CD45RA+), and naive (CCR7+CD45RA+). Then, CM, EM, and TEMRA CD8+ T cells are separated into CD28+ and CD28-. Each one of these CD8+ T cells is first separated into CCR6- and CCR6+, then into Th1 (CCR6-CCR4-CRTH2-), Th2-A (CCR6-CCR4+CRTH2-), Th2-B (CCR6-CCR4+CRTH2+), CCR6-CCR4-CRTH2+, Th1* (CCR6+CCR4-CRTH2-), Th17 (CCR6-CCR4+CRTH2-), CCR6+CCR4+CRTH2+, and CCR6+CCR4-CRTH2+ cells. C.CD3+ T cells, CD3+CD4+ cells are first identified, then into CM (CCR7+CD45RA-), EM (CCR7-CD45RA-), TEMRA (CCR7-CD45RA+), and naive (CCR7+CD45RA+). CM, EM, and TEMRA CD4+ T cells are then separated into CD28+CD27+, CD28+CD27-, and CD28-.Each one of these CD4+ T cells was first separated into CCR6- and CCR6+, then into Th1 (CCR6-CCR4-CRTH2-), Th2-A (CCR6-CCR4+CRTH2-), Th2-B (CCR6-CCR4+CRTH2+), CCR6-CCR4-CRTH2+, Th1* (CCR6+CCR4-CRTH2-), Th17 (CCR6-CCR4+CRTH2-), CCR6+CCR4+CRTH2+, and CCR6+CCR4-CRTH2+ cells. SPHERO™ AccuCount Particles were used to determine absolute counts. Antibodies: anti-CD3 AF700, anti-CD4 PE TR, anti-CD8 BV510, anti-CD45RA BV711, anti-CCR7 BV421, anti-CD27 APC Cy7, anti-CD28 PE Cy7, anti-CCR4 APC, anti-CRTh2 PE, anti-CCR6 BV785, anti-CD19 PerCPCy5.5, anti-IgD BV605, and anti-CD138 FITC. [Figure 1C-1]Figure 1. Flow cytometry gating strategy. (A-C) Doublets are first excluded, followed by identification of lymphocytes by size. A. CD3- are identified, and among these, plasma cells (CD19-CD138+), plasmablasts (CD19+CD138+), B cells (CD19+CD138-), and CD19-CD138- cells are identified. Among B cells, naive B cells (IgD+CD27-), unswitched memory B cells (IgD+CD27+), switched memory B cells (IgD-CD27+), and double negative (IgD-CD27-) B cell subsets are also identified. B. In CD3+ T cells, CD3+CD8+ cells are first identified and then separated into CM (CCR7+CD45RA-), EM (CCR7-CD45RA-), TEMRA (CCR7-CD45RA+), and naive (CCR7+CD45RA+). Then, CM, EM, and TEMRA CD8+ T cells are separated into CD28+ and CD28-. Each one of these CD8+ T cells is first separated into CCR6- and CCR6+, then into Th1 (CCR6-CCR4-CRTH2-), Th2-A (CCR6-CCR4+CRTH2-), Th2-B (CCR6-CCR4+CRTH2+), CCR6-CCR4-CRTH2+, Th1* (CCR6+CCR4-CRTH2-), Th17 (CCR6-CCR4+CRTH2-), CCR6+CCR4+CRTH2+, and CCR6+CCR4-CRTH2+ cells. C.CD3+ T cells, CD3+CD4+ cells are first identified, then into CM (CCR7+CD45RA-), EM (CCR7-CD45RA-), TEMRA (CCR7-CD45RA+), and naive (CCR7+CD45RA+). CM, EM, and TEMRA CD4+ T cells are then separated into CD28+CD27+, CD28+CD27-, and CD28-.Each one of these CD4+ T cells was first separated into CCR6- and CCR6+, then into Th1 (CCR6-CCR4-CRTH2-), Th2-A (CCR6-CCR4+CRTH2-), Th2-B (CCR6-CCR4+CRTH2+), CCR6-CCR4-CRTH2+, Th1* (CCR6+CCR4-CRTH2-), Th17 (CCR6-CCR4+CRTH2-), CCR6+CCR4+CRTH2+, and CCR6+CCR4-CRTH2+ cells. SPHERO™ AccuCount Particles were used to determine absolute counts. Antibodies: anti-CD3 AF700, anti-CD4 PE TR, anti-CD8 BV510, anti-CD45RA BV711, anti-CCR7 BV421, anti-CD27 APC Cy7, anti-CD28 PE Cy7, anti-CCR4 APC, anti-CRTh2 PE, anti-CCR6 BV785, anti-CD19 PerCPCy5.5, anti-IgD BV605, and anti-CD138 FITC. [Figure 1C-2]Figure 1. Flow cytometry gating strategy. (A-C) Doublets are first excluded, followed by identification of lymphocytes by size. A. CD3- are identified, and among these, plasma cells (CD19-CD138+), plasmablasts (CD19+CD138+), B cells (CD19+CD138-), and CD19-CD138- cells are identified. Among B cells, naive B cells (IgD+CD27-), unswitched memory B cells (IgD+CD27+), switched memory B cells (IgD-CD27+), and double negative (IgD-CD27-) B cell subsets are also identified. B. In CD3+ T cells, CD3+CD8+ cells are first identified and then separated into CM (CCR7+CD45RA-), EM (CCR7-CD45RA-), TEMRA (CCR7-CD45RA+), and naive (CCR7+CD45RA+). Then, CM, EM, and TEMRA CD8+ T cells are separated into CD28+ and CD28-. Each one of these CD8+ T cells is first separated into CCR6- and CCR6+, then into Th1 (CCR6-CCR4-CRTH2-), Th2-A (CCR6-CCR4+CRTH2-), Th2-B (CCR6-CCR4+CRTH2+), CCR6-CCR4-CRTH2+, Th1* (CCR6+CCR4-CRTH2-), Th17 (CCR6-CCR4+CRTH2-), CCR6+CCR4+CRTH2+, and CCR6+CCR4-CRTH2+ cells. C.CD3+ T cells, CD3+CD4+ cells are first identified, then into CM (CCR7+CD45RA-), EM (CCR7-CD45RA-), TEMRA (CCR7-CD45RA+), and naive (CCR7+CD45RA+). CM, EM, and TEMRA CD4+ T cells are then separated into CD28+CD27+, CD28+CD27-, and CD28-.Each one of these CD4+ T cells was first separated into CCR6- and CCR6+, then into Th1 (CCR6-CCR4-CRTH2-), Th2-A (CCR6-CCR4+CRTH2-), Th2-B (CCR6-CCR4+CRTH2+), CCR6-CCR4-CRTH2+, Th1* (CCR6+CCR4-CRTH2-), Th17 (CCR6-CCR4+CRTH2-), CCR6+CCR4+CRTH2+, and CCR6+CCR4-CRTH2+ cells. SPHERO™ AccuCount Particles were used to determine absolute counts. Antibodies: anti-CD3 AF700, anti-CD4 PE TR, anti-CD8 BV510, anti-CD45RA BV711, anti-CCR7 BV421, anti-CD27 APC Cy7, anti-CD28 PE Cy7, anti-CCR4 APC, anti-CRTh2 PE, anti-CCR6 BV785, anti-CD19 PerCPCy5.5, anti-IgD BV605, and anti-CD138 FITC. [Figure 1C-3]Figure 1. Flow cytometry gating strategy. (A-C) Doublets are first excluded, followed by identification of lymphocytes by size. A. CD3- are identified, and among these, plasma cells (CD19-CD138+), plasmablasts (CD19+CD138+), B cells (CD19+CD138-), and CD19-CD138- cells are identified. Among B cells, naive B cells (IgD+CD27-), unswitched memory B cells (IgD+CD27+), switched memory B cells (IgD-CD27+), and double negative (IgD-CD27-) B cell subsets are also identified. B. In CD3+ T cells, CD3+CD8+ cells are first identified and then separated into CM (CCR7+CD45RA-), EM (CCR7-CD45RA-), TEMRA (CCR7-CD45RA+), and naive (CCR7+CD45RA+). Then, CM, EM, and TEMRA CD8+ T cells are separated into CD28+ and CD28-. Each one of these CD8+ T cells is first separated into CCR6- and CCR6+, then into Th1 (CCR6-CCR4-CRTH2-), Th2-A (CCR6-CCR4+CRTH2-), Th2-B (CCR6-CCR4+CRTH2+), CCR6-CCR4-CRTH2+, Th1* (CCR6+CCR4-CRTH2-), Th17 (CCR6-CCR4+CRTH2-), CCR6+CCR4+CRTH2+, and CCR6+CCR4-CRTH2+ cells. C.CD3+ T cells, CD3+CD4+ cells are first identified, then into CM (CCR7+CD45RA-), EM (CCR7-CD45RA-), TEMRA (CCR7-CD45RA+), and naive (CCR7+CD45RA+). CM, EM, and TEMRA CD4+ T cells are then separated into CD28+CD27+, CD28+CD27-, and CD28-.Each one of these CD4+ T cells was first separated into CCR6- and CCR6+, then into Th1 (CCR6-CCR4-CRTH2-), Th2-A (CCR6-CCR4+CRTH2-), Th2-B (CCR6-CCR4+CRTH2+), CCR6-CCR4-CRTH2+, Th1* (CCR6+CCR4-CRTH2-), Th17 (CCR6-CCR4+CRTH2-), CCR6+CCR4+CRTH2+, and CCR6+CCR4-CRTH2+ cells. SPHERO™ AccuCount Particles were used to determine absolute counts. Antibodies: anti-CD3 AF700, anti-CD4 PE TR, anti-CD8 BV510, anti-CD45RA BV711, anti-CCR7 BV421, anti-CD27 APC Cy7, anti-CD28 PE Cy7, anti-CCR4 APC, anti-CRTh2 PE, anti-CCR6 BV785, anti-CD19 PerCPCy5.5, anti-IgD BV605, and anti-CD138 FITC.

[0061] [Figure 2A]Figure 2. Recognition of GDP-L-FS and myelin-derived peptides by CSF-infiltrating CD4+ T cells from MS patients. A+B. Proliferative responses expressed as stimulation index (SI) and IFN-γ release expressed as (pg / ml) of PHA-expanded CSF-infiltrating CD4+ T cells to GDP-L-FS, myelin (MBP, MOG(1-20), MOG(35-55), PLP), and CEF peptides presented by autologous PBMC. Each dot represents one well. Each peptide was tested in quadruplicate (4 wells) in 105 MS patients (420 wells in total per peptide). Dotted lines indicate thresholds for positivity (SI ≥ 2 for proliferation and ≥ 20 pg / ml IFN-γ for IFN-γ release). Peptide responses were compared using the Kruskal-Wallis test. Statistical significance of all comparisons is indicated (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001). C. Ratio of % of positive wells with IFN-γ release and proliferation for each peptide. D. Correlation between SI and IFN-γ release (pg / ml) for GDP-L-FS, MBP, MOG(1-20), MOG(35-55), and PLP(139-154) peptides. Spearman r was used to test linear correlation between variables. R values ​​as well as p values ​​are shown. [Figure 2B]Figure 2. Recognition of GDP-L-FS and myelin-derived peptides by CSF-infiltrating CD4+ T cells from MS patients. A+B. Proliferative responses expressed as stimulation index (SI) and IFN-γ release expressed as (pg / ml) of PHA-expanded CSF-infiltrating CD4+ T cells to GDP-L-FS, myelin (MBP, MOG(1-20), MOG(35-55), PLP), and CEF peptides presented by autologous PBMC. Each dot represents one well. Each peptide was tested in quadruplicate (4 wells) in 105 MS patients (420 wells in total per peptide). Dotted lines indicate thresholds for positivity (SI ≥ 2 for proliferation and ≥ 20 pg / ml IFN-γ for IFN-γ release). Peptide responses were compared using the Kruskal-Wallis test. Statistical significance of all comparisons is indicated (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001). C. Ratio of % of positive wells with IFN-γ release and proliferation for each peptide. D. Correlation between SI and IFN-γ release (pg / ml) for GDP-L-FS, MBP, MOG(1-20), MOG(35-55), and PLP(139-154) peptides. Spearman r was used to test linear correlation between variables. R values ​​as well as p values ​​are shown. [Figure 2C]Figure 2. Recognition of GDP-L-FS and myelin-derived peptides by CSF-infiltrating CD4+ T cells from MS patients. A+B. Proliferative responses expressed as stimulation index (SI) and IFN-γ release expressed as (pg / ml) of PHA-expanded CSF-infiltrating CD4+ T cells to GDP-L-FS, myelin (MBP, MOG(1-20), MOG(35-55), PLP), and CEF peptides presented by autologous PBMC. Each dot represents one well. Each peptide was tested in quadruplicate (4 wells) in 105 MS patients (420 wells in total per peptide). Dotted lines indicate thresholds for positivity (SI ≥ 2 for proliferation and ≥ 20 pg / ml IFN-γ for IFN-γ release). Peptide responses were compared using the Kruskal-Wallis test. Statistical significance of all comparisons is indicated (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001). C. Ratio of % of positive wells with IFN-γ release and proliferation for each peptide. D. Correlation between SI and IFN-γ release (pg / ml) for GDP-L-FS, MBP, MOG(1-20), MOG(35-55), and PLP(139-154) peptides. Spearman r was used to test linear correlation between variables. R values ​​as well as p values ​​are shown. [Figure 2D]Figure 2. Recognition of GDP-L-FS and myelin-derived peptides by CSF-infiltrating CD4+ T cells from MS patients. A+B. Proliferative responses expressed as stimulation index (SI) and IFN-γ release expressed as (pg / ml) of PHA-expanded CSF-infiltrating CD4+ T cells to GDP-L-FS, myelin (MBP, MOG(1-20), MOG(35-55), PLP), and CEF peptides presented by autologous PBMC. Each dot represents one well. Each peptide was tested in quadruplicate (4 wells) in 105 MS patients (420 wells in total per peptide). Dotted lines indicate thresholds for positivity (SI ≥ 2 for proliferation and ≥ 20 pg / ml IFN-γ for IFN-γ release). Peptide responses were compared using the Kruskal-Wallis test. Statistical significance of all comparisons is indicated (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001). C. Ratio of % of positive wells with IFN-γ release and proliferation for each peptide. D. Correlation between SI and IFN-γ release (pg / ml) for GDP-L-FS, MBP, MOG(1-20), MOG(35-55), and PLP(139-154) peptides. Spearman r was used to test linear correlation between variables. R values ​​as well as p values ​​are shown.

[0062] [Figure 3A] Figure 3. Identification of GDP-L-FS and Myelin Responder Patients. Checkerboard graphs illustrating the response of each MS patient to individual peptides. Filled and shaded cells are positive responses in proliferation (3A) and IFN-γ release (3B). Non-responders are shown as 3C (proliferation) and 3D (IFN-γ release). The numbers of GDP-L-FS responders, MBP responders, MOG(35-55) responders, and non-responders are shown. [Figure 3B]Figure 3. Identification of GDP-L-FS and Myelin Responder Patients. Checkerboard graphs illustrating the response of each MS patient to individual peptides. Filled and shaded cells are positive responses in proliferation (3A) and IFN-γ release (3B). Non-responders are shown as 3C (proliferation) and 3D (IFN-γ release). The numbers of GDP-L-FS responders, MBP responders, MOG(35-55) responders, and non-responders are shown. [Figure 3C-D] Figure 3. Identification of GDP-L-FS and Myelin Responder Patients. Checkerboard graphs illustrating the response of each MS patient to individual peptides. Filled and shaded cells are positive responses in proliferation (3A) and IFN-γ release (3B). Non-responders are shown as 3C (proliferation) and 3D (IFN-γ release). The numbers of GDP-L-FS responders, MBP responders, MOG(35-55) responders, and non-responders are shown.

[0063] [Figure 4A] Figure 4. Differences in CSF-infiltrating and circulating lymphocytes in GDP-L-FS responders, MOG(35-55) responders, and non-responders. A. Dot plots showing CD28 and CD27 expression on CSF-infiltrating and peripherally circulating EM CD4+ cells from GDP-L-FS responders and non-responders. Percentages of EM CD27- cells are shown. B-C. Frequency and absolute numbers of CSF-infiltrating (B) and peripherally circulating (C) EM CD27- and EM CD27- Th1 cells in GDP-L-FS responders, MOG35-55 responders, and non-responders. Cell numbers were determined using SPHERO™ AccuCount Particles. Each dot corresponds to one patient and bars indicate the mean. Patients were compared using the Kruskal-Wallis test. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 4B]Figure 4. Differences in CSF-infiltrating and circulating lymphocytes in GDP-L-FS responders, MOG(35-55) responders, and non-responders. A. Dot plots showing CD28 and CD27 expression on CSF-infiltrating and peripherally circulating EM CD4+ cells from GDP-L-FS responders and non-responders. Percentages of EM CD27- cells are shown. B-C. Frequency and absolute numbers of CSF-infiltrating (B) and peripherally circulating (C) EM CD27- and EM CD27- Th1 cells in GDP-L-FS responders, MOG35-55 responders, and non-responders. Cell numbers were determined using SPHERO™ AccuCount Particles. Each dot corresponds to one patient and bars indicate the mean. Patients were compared using the Kruskal-Wallis test. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 4C] Figure 4. Differences in CSF-infiltrating and circulating lymphocytes in GDP-L-FS responders, MOG(35-55) responders, and non-responders. A. Dot plots showing CD28 and CD27 expression on CSF-infiltrating and peripherally circulating EM CD4+ cells from GDP-L-FS responders and non-responders. Percentages of EM CD27- cells are shown. B-C. Frequency and absolute numbers of CSF-infiltrating (B) and peripherally circulating (C) EM CD27- and EM CD27- Th1 cells in GDP-L-FS responders, MOG35-55 responders, and non-responders. Cell numbers were determined using SPHERO™ AccuCount Particles. Each dot corresponds to one patient and bars indicate the mean. Patients were compared using the Kruskal-Wallis test. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated.

[0064] [Figure 5A]Figure 5. Ex-vivo flow cytometric immunophenotyping of CSF-infiltrating and circulating T lymphocytes. Frequencies of CSF-infiltrating cells (A–B) and peripheral circulating cells (C–F) as well as central memory (CM, CCR7+CD45RA-), effector memory (EM, CCR7-CD45RA-), and TEMRA (CCR7-CD45RA+) CD4+ T cell subsets expressing CD28+CD27+, CD28+CD27-, and CD28-, and EM with functional phenotypes based on chemokine receptor expression: Actual numbers of CD28+CD27-CD4+ T cells: Th2A (CCR6-CCR4+CRth2-), Th2B (CCR6-CCR4+CRth2+), CCR6-CCR4-CRth2+, Th1 (CCR6-CCR4-CRTh2-), Th17 (CCR6+CCR4+CRth2-), CCR6+CCR4+CRth2+, CCR6+CCR4-CRth2+, and Th1* (CCR6+CCR4-CRth2-). Each dot in the graph corresponds to one patient and the bars represent the mean. Cell counts were determined using SPHERO™ AccuCount Particles. Kruskal-Wallis test was used to compare GDP-L-FS responders, MOG35-55 responders, and non-responders. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 5B]Figure 5. Ex-vivo flow cytometric immunophenotyping of CSF-infiltrating and circulating T lymphocytes. Frequencies of CSF-infiltrating cells (A–B) and peripheral circulating cells (C–F) as well as central memory (CM, CCR7+CD45RA-), effector memory (EM, CCR7-CD45RA-), and TEMRA (CCR7-CD45RA+) CD4+ T cell subsets expressing CD28+CD27+, CD28+CD27-, and CD28-, and EM with functional phenotypes based on chemokine receptor expression: Actual numbers of CD28+CD27-CD4+ T cells: Th2A (CCR6-CCR4+CRth2-), Th2B (CCR6-CCR4+CRth2+), CCR6-CCR4-CRth2+, Th1 (CCR6-CCR4-CRTh2-), Th17 (CCR6+CCR4+CRth2-), CCR6+CCR4+CRth2+, CCR6+CCR4-CRth2+, and Th1* (CCR6+CCR4-CRth2-). Each dot in the graph corresponds to one patient and the bars represent the mean. Cell counts were determined using SPHERO™ AccuCount Particles. Kruskal-Wallis test was used to compare GDP-L-FS responders, MOG35-55 responders, and non-responders. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 5C]Figure 5. Ex-vivo flow cytometric immunophenotyping of CSF-infiltrating and circulating T lymphocytes. Frequencies of CSF-infiltrating cells (A–B) and peripheral circulating cells (C–F) as well as central memory (CM, CCR7+CD45RA-), effector memory (EM, CCR7-CD45RA-), and TEMRA (CCR7-CD45RA+) CD4+ T cell subsets expressing CD28+CD27+, CD28+CD27-, and CD28-, and EM with functional phenotypes based on chemokine receptor expression: Actual numbers of CD28+CD27-CD4+ T cells: Th2A (CCR6-CCR4+CRth2-), Th2B (CCR6-CCR4+CRth2+), CCR6-CCR4-CRth2+, Th1 (CCR6-CCR4-CRTh2-), Th17 (CCR6+CCR4+CRth2-), CCR6+CCR4+CRth2+, CCR6+CCR4-CRth2+, and Th1* (CCR6+CCR4-CRth2-). Each dot in the graph corresponds to one patient and the bars represent the mean. Cell counts were determined using SPHERO™ AccuCount Particles. Kruskal-Wallis test was used to compare GDP-L-FS responders, MOG35-55 responders, and non-responders. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 5D]Figure 5. Ex-vivo flow cytometric immunophenotyping of CSF-infiltrating and circulating T lymphocytes. Frequencies of CSF-infiltrating cells (A–B) and peripheral circulating cells (C–F) as well as central memory (CM, CCR7+CD45RA-), effector memory (EM, CCR7-CD45RA-), and TEMRA (CCR7-CD45RA+) CD4+ T cell subsets expressing CD28+CD27+, CD28+CD27-, and CD28-, and EM with functional phenotypes based on chemokine receptor expression: Actual numbers of CD28+CD27-CD4+ T cells: Th2A (CCR6-CCR4+CRth2-), Th2B (CCR6-CCR4+CRth2+), CCR6-CCR4-CRth2+, Th1 (CCR6-CCR4-CRTh2-), Th17 (CCR6+CCR4+CRth2-), CCR6+CCR4+CRth2+, CCR6+CCR4-CRth2+, and Th1* (CCR6+CCR4-CRth2-). Each dot in the graph corresponds to one patient and the bars represent the mean. Cell counts were determined using SPHERO™ AccuCount Particles. Kruskal-Wallis test was used to compare GDP-L-FS responders, MOG35-55 responders, and non-responders. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 5E]Figure 5. Ex-vivo flow cytometric immunophenotyping of CSF-infiltrating and circulating T lymphocytes. Frequencies of CSF-infiltrating cells (A–B) and peripheral circulating cells (C–F) as well as central memory (CM, CCR7+CD45RA-), effector memory (EM, CCR7-CD45RA-), and TEMRA (CCR7-CD45RA+) CD4+ T cell subsets expressing CD28+CD27+, CD28+CD27-, and CD28-, and EM with functional phenotypes based on chemokine receptor expression: Actual numbers of CD28+CD27-CD4+ T cells: Th2A (CCR6-CCR4+CRth2-), Th2B (CCR6-CCR4+CRth2+), CCR6-CCR4-CRth2+, Th1 (CCR6-CCR4-CRTh2-), Th17 (CCR6+CCR4+CRth2-), CCR6+CCR4+CRth2+, CCR6+CCR4-CRth2+, and Th1* (CCR6+CCR4-CRth2-). Each dot in the graph corresponds to one patient and the bars represent the mean. Cell counts were determined using SPHERO™ AccuCount Particles. Kruskal-Wallis test was used to compare GDP-L-FS responders, MOG35-55 responders, and non-responders. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 5F]Figure 5. Ex-vivo flow cytometric immunophenotyping of CSF-infiltrating and circulating T lymphocytes. Frequencies of CSF-infiltrating cells (A–B) and peripheral circulating cells (C–F) as well as central memory (CM, CCR7+CD45RA-), effector memory (EM, CCR7-CD45RA-), and TEMRA (CCR7-CD45RA+) CD4+ T cell subsets expressing CD28+CD27+, CD28+CD27-, and CD28-, and EM with functional phenotypes based on chemokine receptor expression: Actual numbers of CD28+CD27-CD4+ T cells: Th2A (CCR6-CCR4+CRth2-), Th2B (CCR6-CCR4+CRth2+), CCR6-CCR4-CRth2+, Th1 (CCR6-CCR4-CRTh2-), Th17 (CCR6+CCR4+CRth2-), CCR6+CCR4+CRth2+, CCR6+CCR4-CRth2+, and Th1* (CCR6+CCR4-CRth2-). Each dot in the graph corresponds to one patient and the bars represent the mean. Cell counts were determined using SPHERO™ AccuCount Particles. Kruskal-Wallis test was used to compare GDP-L-FS responders, MOG35-55 responders, and non-responders. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated.

[0065] [Figure 6A]Figure 6. Purification and transcriptome analysis of EM CD27+ / CD27- cells. A. Gating strategy used to isolate EM CD28+CD4+ T cells expressing or not expressing CD27 from four GDP-L-FS responder MS patients and four HDs. The frequency of EM CD28+CD4+ T cells expressing or not expressing CD27 before and after cell sorting from one representative GDP-L-FS responder MS patient and one HD is shown. The Mann-Whitney test was used to compare the frequency of EM CD27 cells in GDP-L-FS responder patients and HDs, and statistical significance (*p<0.05) is indicated. B. Heatmap showing row-wise z-scores of 145 differentially expressed transcripts identified by RNA-seq analysis and pairwise comparison (Log2 ratio >0.5, p<0.001) of EM CD27- from GDP-L-FS responders (columns 5-8) to EM CD27- from HDs (columns E-H). Heatmaps also show the rowwise z-scores of these 145 transcripts in EM CD27+ cells from GDP-L-FS responders (columns 1-4) and HD (columns A-D). Rowwise z-scores of selected transcripts associated with cytotoxicity, Th1, and other Th subsets are detailed. In bold are genes that were also identified as differentially expressed by RNA-seq analysis and pairwise comparisons (Log2 ratio >0.5, p<0.001) between EM CD27- and EM CD27+ in GDP-L-FS patients. [Figure 6B]Figure 6. Purification and transcriptome analysis of EM CD27+ / CD27- cells. A. Gating strategy used to isolate EM CD28+CD4+ T cells expressing or not expressing CD27 from four GDP-L-FS responder MS patients and four HDs. The frequency of EM CD28+CD4+ T cells expressing or not expressing CD27 before and after cell sorting from one representative GDP-L-FS responder MS patient and one HD is shown. The Mann-Whitney test was used to compare the frequency of EM CD27 cells in GDP-L-FS responder patients and HDs, and statistical significance (*p<0.05) is indicated. B. Heatmap showing row-wise z-scores of 145 differentially expressed transcripts identified by RNA-seq analysis and pairwise comparison (Log2 ratio >0.5, p<0.001) of EM CD27- from GDP-L-FS responders (columns 5-8) to EM CD27- from HDs (columns E-H). Heatmaps also show the rowwise z-scores of these 145 transcripts in EM CD27+ cells from GDP-L-FS responders (columns 1-4) and HD (columns A-D). Rowwise z-scores of selected transcripts associated with cytotoxicity, Th1, and other Th subsets are detailed. In bold are genes that were also identified as differentially expressed by RNA-seq analysis and pairwise comparisons (Log2 ratio >0.5, p<0.001) between EM CD27- and EM CD27+ in GDP-L-FS patients.

[0066] [Figure 7A]Figure 7. Transcriptome analysis of EM CD27- and EM CD27+CD4+ T cells. A. Heatmap shows row-wise z-scores of 265 transcripts that were differentially expressed between EM CD27+ cells (columns 1-4) and EM CD27- cells (columns 5-8) from four GDP-L-FS responder patients (Log2 ratio >0.5, p<0.001). Also shown are the z-scores of these genes in HD-derived EM CD27+ cells (columns A-D) and EM CD27- cells (columns E-H). Detail the z-scores of selected genes related to cytotoxicity, Th1, and other Th subsets. In bold are genes that were also differentially expressed between EM CD27- from GDP-L-FS responders and EM CD27- from HD (Log2 ratio >0.5, p<0.001). B. Distribution of log2 (Fragments Per Kilobase per Million (FPKM)+0.1) gene expression of Th1 / cytotoxicity genes and genes associated with other Th subsets in EM CD27+ and CD27- cells from GDP-L-FS responders and HD. [Figure 7B]Figure 7. Transcriptome analysis of EM CD27- and EM CD27+CD4+ T cells. A. Heatmap shows row-wise z-scores of 265 transcripts that were differentially expressed between EM CD27+ cells (columns 1-4) and EM CD27- cells (columns 5-8) from four GDP-L-FS responder patients (Log2 ratio >0.5, p<0.001). Also shown are the z-scores of these genes in HD-derived EM CD27+ cells (columns A-D) and EM CD27- cells (columns E-H). Detail the z-scores of selected genes related to cytotoxicity, Th1, and other Th subsets. In bold are genes that were also differentially expressed between EM CD27- from GDP-L-FS responders and EM CD27- from HD (Log2 ratio >0.5, p<0.001). B. Distribution of log2 (Fragments Per Kilobase per Million (FPKM)+0.1) gene expression of Th1 / cytotoxicity genes and genes associated with other Th subsets in EM CD27+ and CD27- cells from GDP-L-FS responders and HD.

[0067] [Figure 8A-1]Figure 8. Characterization of GDP-L-FS and MOG(35-55) specific responders. A+B. Top graph: Cytokines released by CSF infiltrating CD4+ T cells from GDP-L-FS and MOG(35-55) responders after stimulation with specific peptides (GDP-L-FS and MOG(35-55)) presented by autologous PBMC. Four wells were pooled for each patient. Bottom graph: Cytokines present in the CSF of GDP-L-FS and MOG(35-55) responders. Cytokines are expressed as pg / ml. C. Top graph: Proliferative response of CSF infiltrating CD4+ T cells from GDP-L-FS and MOG(35-55) responders after stimulation with specific peptides presented by autologous PBMC or anti-CD3, anti-CD28, anti-CD2 stimulatory beads. Proliferative response is expressed as SI. Bottom graph: Frequency of CSF-infiltrating and blood-circulating naive (IgD+CD27-) B (CD19+CD138-) cells in GDP-L-FS and MOG35-55 responders. Each dot in the graph corresponds to one patient, and the bars show the mean. Mann-Whitney test was used to compare GDP-L-FS and MOG(35-55) responders. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 8A-2]Figure 8. Characterization of GDP-L-FS and MOG(35-55) specific responders. A+B. Top graph: Cytokines released by CSF infiltrating CD4+ T cells from GDP-L-FS and MOG(35-55) responders after stimulation with specific peptides (GDP-L-FS and MOG(35-55)) presented by autologous PBMC. Four wells were pooled for each patient. Bottom graph: Cytokines present in the CSF of GDP-L-FS and MOG(35-55) responders. Cytokines are expressed as pg / ml. C. Top graph: Proliferative response of CSF infiltrating CD4+ T cells from GDP-L-FS and MOG(35-55) responders after stimulation with specific peptides presented by autologous PBMC or anti-CD3, anti-CD28, anti-CD2 stimulatory beads. Proliferative response is expressed as SI. Bottom graph: Frequency of CSF-infiltrating and blood-circulating naive (IgD+CD27-) B (CD19+CD138-) cells in GDP-L-FS and MOG35-55 responders. Each dot in the graph corresponds to one patient, and the bars show the mean. Mann-Whitney test was used to compare GDP-L-FS and MOG(35-55) responders. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 8B-1]Figure 8. Characterization of GDP-L-FS and MOG(35-55) specific responders. A+B. Top graph: Cytokines released by CSF infiltrating CD4+ T cells from GDP-L-FS and MOG(35-55) responders after stimulation with specific peptides (GDP-L-FS and MOG(35-55)) presented by autologous PBMC. Four wells were pooled for each patient. Bottom graph: Cytokines present in the CSF of GDP-L-FS and MOG(35-55) responders. Cytokines are expressed as pg / ml. C. Top graph: Proliferative response of CSF infiltrating CD4+ T cells from GDP-L-FS and MOG(35-55) responders after stimulation with specific peptides presented by autologous PBMC or anti-CD3, anti-CD28, anti-CD2 stimulatory beads. Proliferative response is expressed as SI. Bottom graph: Frequency of CSF-infiltrating and blood-circulating naive (IgD+CD27-) B (CD19+CD138-) cells in GDP-L-FS and MOG35-55 responders. Each dot in the graph corresponds to one patient, and the bars show the mean. Mann-Whitney test was used to compare GDP-L-FS and MOG(35-55) responders. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 8B-2]Figure 8. Characterization of GDP-L-FS and MOG(35-55) specific responders. A+B. Top graph: Cytokines released by CSF infiltrating CD4+ T cells from GDP-L-FS and MOG(35-55) responders after stimulation with specific peptides (GDP-L-FS and MOG(35-55)) presented by autologous PBMC. Four wells were pooled for each patient. Bottom graph: Cytokines present in the CSF of GDP-L-FS and MOG(35-55) responders. Cytokines are expressed as pg / ml. C. Top graph: Proliferative response of CSF infiltrating CD4+ T cells from GDP-L-FS and MOG(35-55) responders after stimulation with specific peptides presented by autologous PBMC or anti-CD3, anti-CD28, anti-CD2 stimulatory beads. Proliferative response is expressed as SI. Bottom graph: Frequency of CSF-infiltrating and blood-circulating naive (IgD+CD27-) B (CD19+CD138-) cells in GDP-L-FS and MOG35-55 responders. Each dot in the graph corresponds to one patient, and the bars show the mean. Mann-Whitney test was used to compare GDP-L-FS and MOG(35-55) responders. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 8C]Figure 8. Characterization of GDP-L-FS and MOG(35-55) specific responders. A+B. Top graph: Cytokines released by CSF infiltrating CD4+ T cells from GDP-L-FS and MOG(35-55) responders after stimulation with specific peptides (GDP-L-FS and MOG(35-55)) presented by autologous PBMC. Four wells were pooled for each patient. Bottom graph: Cytokines present in the CSF of GDP-L-FS and MOG(35-55) responders. Cytokines are expressed as pg / ml. C. Top graph: Proliferative response of CSF infiltrating CD4+ T cells from GDP-L-FS and MOG(35-55) responders after stimulation with specific peptides presented by autologous PBMC or anti-CD3, anti-CD28, anti-CD2 stimulatory beads. Proliferative response is expressed as SI. Bottom graph: Frequency of CSF-infiltrating and blood-circulating naive (IgD+CD27-) B (CD19+CD138-) cells in GDP-L-FS and MOG35-55 responders. Each dot in the graph corresponds to one patient, and the bars show the mean. Mann-Whitney test was used to compare GDP-L-FS and MOG(35-55) responders. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated.

[0068] [Figure 9A]Figure 9. Further characterization of CSF from patients and controls. A. Cytokines present in the CSF of MOG(35-55) responders and MOGAD patients. Cytokine release is expressed as pg / ml. B-C. The following in GDP-L-FS and MOG(35-55) responders and CP: B, CXCL13, chitinase-3-like protein 1 (CHI3L1), and intrathecal IgG synthesis IgG(loc); C, intrathecal abundance of granulysin, granzyme H (GZMH), granzyme A (GZMA), and neurofilament light chain (NfL). Each dot in the graph corresponds to one patient and the bars show the mean. The Mann-Whitney test was used to compare MOG(35-55) responders and anti-MOG patients, and the Kruskal-Wallis was used to compare GDP-L-FS responders, MOG(35-55) responders, and CP. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated. [Figure 9B-C] Figure 9. Further characterization of CSF from patients and controls. A. Cytokines present in the CSF of MOG(35-55) responders and MOGAD patients. Cytokine release is expressed as pg / ml. B-C. The following in GDP-L-FS and MOG(35-55) responders and CP: B, CXCL13, chitinase-3-like protein 1 (CHI3L1), and intrathecal IgG synthesis IgG(loc); C, intrathecal abundance of granulysin, granzyme H (GZMH), granzyme A (GZMA), and neurofilament light chain (NfL). Each dot in the graph corresponds to one patient and the bars show the mean. The Mann-Whitney test was used to compare MOG(35-55) responders and anti-MOG patients, and the Kruskal-Wallis was used to compare GDP-L-FS responders, MOG(35-55) responders, and CP. Statistical significance (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) is indicated.

[0069] [Figure 10A]Figure 10. HLA class II expression in GDP-L-FS and MOG35-55 responder patients. A. Frequency of patients expressing the DR15 gene (left) and the DRB3*02:02 / 03:01 gene (right) in GDP-L-FS responders, MOG(35-55) responders, non-responders, and two reference cohorts. The number of patients who responded or did not respond to GDP-L-FS and expressed (Y, yes) or did not (N, no) the DRB3*02:02 / 03:01 gene, as well as the P value for Fisher's exact test, are shown. B+C. Responses (proliferation (SI) and IFN-γ release) of CSF-infiltrating CD4+ T cells to GDP-L-FS, MBP, MOG(1-20), MOG(35-55), PLP, and CEF peptides, as well as anti-CD2, anti-CD28, and anti-CD3 stimulatory beads from patients expressing or not expressing the DRB3*02:02 / 03:01 gene. Each dot in the graph corresponds to a single well. The Mann-Whitney test was used to compare patients expressing DRB3*02:02 / 03:01 and other DRs. Statistical significance (***p<0.001 and ****p<0.0001) is indicated. [Figure 10B]Figure 10. HLA class II expression in GDP-L-FS and MOG35-55 responder patients. A. Frequency of patients expressing the DR15 gene (left) and the DRB3*02:02 / 03:01 gene (right) in GDP-L-FS responders, MOG(35-55) responders, non-responders, and two reference cohorts. The number of patients who responded or did not respond to GDP-L-FS and expressed (Y, yes) or did not (N, no) the DRB3*02:02 / 03:01 gene, as well as the P value for Fisher's exact test, are shown. B+C. Responses (proliferation (SI) and IFN-γ release) of CSF-infiltrating CD4+ T cells to GDP-L-FS, MBP, MOG(1-20), MOG(35-55), PLP, and CEF peptides, as well as anti-CD2, anti-CD28, and anti-CD3 stimulatory beads from patients expressing or not expressing the DRB3*02:02 / 03:01 gene. Each dot in the graph corresponds to a single well. The Mann-Whitney test was used to compare patients expressing DRB3*02:02 / 03:01 and other DRs. Statistical significance (***p<0.001 and ****p<0.0001) is indicated. [Figure 10C]Figure 10. HLA class II expression in GDP-L-FS and MOG35-55 responder patients. A. Frequency of patients expressing the DR15 gene (left) and the DRB3*02:02 / 03:01 gene (right) in GDP-L-FS responders, MOG(35-55) responders, non-responders, and two reference cohorts. The number of patients who responded or did not respond to GDP-L-FS and expressed (Y, yes) or did not (N, no) the DRB3*02:02 / 03:01 gene, as well as the P value for Fisher's exact test, are shown. B+C. Responses (proliferation (SI) and IFN-γ release) of CSF-infiltrating CD4+ T cells to GDP-L-FS, MBP, MOG(1-20), MOG(35-55), PLP, and CEF peptides, as well as anti-CD2, anti-CD28, and anti-CD3 stimulatory beads from patients expressing or not expressing the DRB3*02:02 / 03:01 gene. Each dot in the graph corresponds to a single well. The Mann-Whitney test was used to compare patients expressing DRB3*02:02 / 03:01 and other DRs. Statistical significance (***p<0.001 and ****p<0.0001) is indicated.

[0070] [Figure 11A] Figure 11. Characterization of GDP-L-FS and MOG35-55 responder patients. A+B. Monthly distribution of LP and frequency of samples obtained in winter / spring and summer / autumn in GDP-L-FS responders, MOG35-55 responders, and non-responders. The number of patients responding or not responding to GDP-L-FS who obtained LP in winter / spring (Y, yes) or not (N, no) and the P value of Fisher's exact test are shown. C. Total contrast-enhanced T1 lesion count and total flair T2 lesion volume (expressed in mL) in GDP-L-FS and MOG(35-55) responders. Each dot in the graph corresponds to one patient and the bars show the mean. The two groups of patients were compared using a T-test for normally distributed variables. Statistical significance (**p<0.01) is indicated. [Figure 11B]Figure 11. Characterization of GDP-L-FS and MOG35-55 responder patients. A+B. Monthly distribution of LP and frequency of samples obtained in winter / spring and summer / autumn in GDP-L-FS responders, MOG35-55 responders, and non-responders. The number of patients responding or not responding to GDP-L-FS who obtained LP in winter / spring (Y, yes) or not (N, no) and the P value of Fisher's exact test are shown. C. Total contrast-enhanced T1 lesion count and total flair T2 lesion volume (expressed in mL) in GDP-L-FS and MOG(35-55) responders. Each dot in the graph corresponds to one patient and the bars show the mean. The two groups of patients were compared using a T-test for normally distributed variables. Statistical significance (**p<0.01) is indicated. [Figure 11C] Figure 11. Characterization of GDP-L-FS and MOG35-55 responder patients. A+B. Monthly distribution of LP and frequency of samples obtained in winter / spring and summer / autumn in GDP-L-FS responders, MOG35-55 responders, and non-responders. The number of patients responding or not responding to GDP-L-FS who obtained LP in winter / spring (Y, yes) or not (N, no) and the P value of Fisher's exact test are shown. C. Total contrast-enhanced T1 lesion count and total flair T2 lesion volume (expressed in mL) in GDP-L-FS and MOG(35-55) responders. Each dot in the graph corresponds to one patient and the bars show the mean. The two groups of patients were compared using a T-test for normally distributed variables. Statistical significance (**p<0.01) is indicated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] Detailed Description of the Invention A method for stratifying MS patients has been found, which allows to tailor the therapeutic approach specifically for each individual patient (individualized treatment).For example, patients with CD27-Th1CD4+ cells, preferably CCR7-CD45RA-CD27-Th1CD4+ cells, detected in body fluids, particularly blood, preferably peripheral blood, or CSF (obtained beforehand from MS patients) can be selected for tolerization approach.Preferably, the patient's immune system, particularly T cells and / or antibodies in body fluids, are reactive (i.e., responsive) to protein GDP-L-FS, or its fragments, derivatives, and / or splice variants.

[0072] Stratification preferably means classifying MS patients into different groups according to the outcome of the method. For example, the patient subpopulation that can be identified by the method of the present application is characterized by CD27-Th1CD4+ cells in body fluids, in particular in peripheral blood or CSF. In one embodiment, the same patient subpopulation can be further characterized by the responsiveness of T cells and / or antibodies in the body fluids to the protein GDP-L-FS, or its fragments, derivatives, and / or splice variants. In another particular embodiment, this patient subpopulation can be characterized by the following characteristics: - Inflammation and / or neurodegeneration of the central nervous system specifically characterized by Gd-contrast enhanced T1 lesions and / or FLAIR T2 lesions, - higher expression of genes associated with Th1 cells or cytotoxicity and / or genes encoding proinflammatory cytokines such as IL-2 and / or IFN-γ compared to healthy controls, -HLA allotype HLA-DRB3 * 02:02 or DRB3 * 03:01 The compound may be further characterized by one or more of:

[0073] According to one embodiment of the invention, the CD27-Th1CD4+ cells detected in body fluids, in particular in peripheral blood or CSF, are additionally negative for the markers CCR7 and / or CD45RA. In a preferred embodiment, the CD27-Th1CD4+ cells are negative for both the markers CCR7 and CD45RA.

[0074] In a particularly preferred embodiment, the patient's T cells respond to at least one of the peptides defined in SEQ ID NO: 2-6 and SEQ ID NO: 37. In another preferred embodiment, the fragment comprises or consists of any sequence within the sequence defined by SEQ ID NO: 37. The peptides may also be referred to as immunodominant.

[0075] The T cells which respond to the protein GDP-L-FS, or fragments, derivatives and / or splice variants thereof, are preferably CD4+ T cells, more preferably CSF-infiltrating CD4+ T cells.

[0076] T cell response to a particular stimulus can be measured, for example, by T cell proliferation and / or release of cytokines, especially IFN-γ. Those skilled in the art know how to measure proliferation and release / secretion of cytokines (such as IFN-γ). For example, proliferation response can be measured by 3H-thymidine assay. Cytokines can be quantified, for example, by conventional ELISA test or bead-based immunoassay that can simultaneously quantify multiple cytokines using flow cytometer.

[0077] A patient is preferably classified as a responder if it exhibits a positive proliferative response and / or if cytokines (such as IFN-γ) can be detected in response to the stimulus (ie, preferably the peptide).

[0078] The cytoplasmic enzyme GDP-L-fucose synthase converts GDP-4-keto-6-deoxy-D-mannose to GDP-L-fucose, which is then used by fucosyltransferases to fucosylate all oligosaccharides. In mammals, fucosylated glycans play important roles in many biological processes, including transfusion response, host-microbe interactions, cancer pathogenesis, and maintaining a non-inflammatory environment in the brain. In one embodiment, GDP-L-fucose synthase exhibits the enzymatic activity of converting GDP-4-keto-6-deoxy-D-mannose to GDP-L-fucose.

[0079] Protein is intended to mean oligopeptides, polypeptides, and proteins themselves. Protein sequences may be defined by GenBank entries. Protein sequences may also be defined by UniProtKB / Swiss-Prot entries and / or GenPept entries. Entries may be defined by numbers (e.g., accession numbers). Where applicable, database entries include their respective accession numbers (i.e., entry numbers) and version numbers. Proteins may also be defined by any other database known to those skilled in the art. Different isoforms, derivatives, and / or splice variants may exist and are also encompassed by the present invention. Thereby, sequences may differ from known sequences, for example from GenBank or UniProtKB / Swiss-Prot entries.

[0080] Unless expressly stated otherwise, "a" protein or "the" protein of the present invention refers to, for example, a GDP-L-fucose synthase protein.

[0081] In a preferred embodiment, the protein is a human protein and / or the nucleotide sequence and / or gene sequence is a human sequence.

[0082] Splice variants arise from alternative splicing during gene expression. The splice variants of the present invention are preferably immunodominant.

[0083] A fragment is preferably any part of a protein that is shorter, i.e. has fewer amino acids, than the parent protein. A fragment may be a peptide. In one embodiment, the fragment comprises 5-50, preferably 5-20, more preferably 10-15 amino acids, even more preferably 15 amino acids. The fragments of the present invention are preferably immunodominant.

[0084] A derivative of a sequence is preferably defined as an amino acid sequence having at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homology or identity with the corresponding portion of a reference amino acid sequence over the entire length of the sequence. A "corresponding portion" in the sense of the present invention preferably refers to an identical stretch of amino acids of the same parent sequence. For example, if a derivative 100 amino acids in length differs from a stretch of amino acids of SEQ ID NO: 1 (amino acids 1-100 of SEQ ID NO: 1) by 20 amino acids, this particular derivative shares 80% identity with the corresponding portion (i.e., amino acids 1-100 of the reference amino acid sequence (i.e., SEQ ID NO: 1)) over its entire length. Derivatives according to the present invention are preferably immunodominant.

[0085] Amino acid sequence "homology" or "identity" is preferably determined according to the present invention over the entire length of the reference amino acid sequence or over the entire length of the corresponding portion of the reference amino acid sequence to which the sequence for which homology or identity is defined.

[0086] "Identity" is defined as identical amino acids, and "homology" includes identical amino acids and conservative substitutions. Those of skill in the art are aware of conservative substitutions such as: -Aromatic and aromatic (F and W / Y) -Positive and negative charges (R and K / H) -negative charges (E and D) or -Aliphatic (V and L / M / I, or A and S / T).

[0087] The nucleotide sequence that codes for the protein of the present invention or any of its fragments, derivatives or splice variants refers to any coding nucleotide sequence, for example RNA or DNA, particularly mRNA or cDNA.In one embodiment, the nucleotide sequence is a plasmid or any type of vector known to those skilled in the art.In a preferred embodiment, the nucleotide sequence does not include introns, and the gene sequence includes exons and introns.

[0088] In a preferred embodiment, the GDP-L-fucose synthase protein is a) having the amino acid sequence set forth in SEQ ID NO:1; b) has an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1; c) having an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1; d) has an amino acid sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the amino acid sequence set forth in SEQ ID NO:1, and said protein, or a fragment or splice variant thereof, binds to autologous HLA alleles, is recognized by T cells, and / or is recognized by antibodies that bind to or recognize the amino acid sequence set forth in SEQ ID NO:1 or a fragment thereof; or e) TSTA3 gene, in particular encoded by the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11 or encoded by a gene that is at least 80%, preferably at least 90%, even more preferably at least 95% identical to the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11.

[0089] Binding to autologous HLA alleles, recognition by T cells, and / or recognition by antibodies may indicate immunodominance of a protein or a fragment or splice variant thereof, and immunodominance can be tested as disclosed below.

[0090] In one embodiment, the fragment comprises 5 to 50, preferably 5 to 20, more preferably 10 to 15, and even more preferably 15 amino acids.

[0091] In another embodiment, the fragment is a) is at least 85%, preferably at least 90%, more preferably at least 95% identical to the respective corresponding amino acid sequence; b) are at least 70%, preferably at least 80%, more preferably at least 90% homologous to their respective corresponding amino acid sequences; or c) are at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to their respective corresponding amino acid sequences, bind to autologous HLA alleles, are recognized by T cells, and / or are recognized by antibodies that bind to or recognize the respective amino acid sequences.

[0092] "Respective corresponding amino acid sequences" refer to the respective fragments of the corresponding amino acid sequence (i.e. SEQ ID NO: 1) that have the same length as the homologous fragment (see also the definition of "corresponding portion" above). These identical and / or homologous fragments may contain 5 to 50, preferably 5 to 20, more preferably 10 to 15, even more preferably 15 amino acids. The identity and / or homology is determined over the entire length of the respective fragment. In other words, "corresponding amino acid sequences" refer to the invariant sequences (i.e. if a fragment of the sequence set forth in SEQ ID NO: 1 is 85% identical to the respective corresponding amino acids, the fragment is 85% identical (over the entire length of the fragment) to the invariant fragment "excised" (i.e. obtained or copied directly from SEQ ID NO: 1).

[0093] Thus, in one embodiment, the protein GDP-L-fucose synthase has an amino acid sequence with a certain homology (at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%) to the respective indicated sequences set forth in SEQ ID NOs, with the further requirement that said protein or a fragment or splice variant thereof binds to autologous HLA alleles and is recognized by T cells and / or is recognized by antibodies that bind to or recognize the amino acid sequence set forth in the respective SEQ ID NOs or a fragment thereof. In another embodiment, the protein or a fragment or splice variant thereof binds to autologous HLA alleles and is recognized by T cells that bind to or recognize the amino acid sequence set forth in the respective SEQ ID NOs or a fragment thereof.

[0094] Assays for measuring and / or predicting binding to autologous HLA alleles, recognition by T cells, or recognition by antibodies are well known to those skilled in the art. Binding of peptides to HLA alleles can be predicted, for example, using the widely accepted NetMHCII (http: / / www.cbs.dtu.dk / services / NetMHCII / ) or IEDB (http: / / www.iedb.org / ) in silico peptide binding prediction algorithms. T cell recognition can be measured, for example, by T cell proliferation assay, for example by measuring incorporated radioactivity. Binding of peptides and / or proteins to antibodies can be measured by standard assays known to those skilled in the art (for example, by ELISA). Binding to autologous HLA alleles, recognition by T cells, or recognition by antibodies can indicate the immunodominance of peptides or proteins. Immunodominance can also be tested as disclosed below.

[0095] In a preferred embodiment, the peptide used for the treatment of the present invention comprises a fragment of GDP-L-fucose synthase and comprises at least one sequence selected from the group consisting of SEQ ID NO: 2 to 6 and SEQ ID NO: 37. In a particularly preferred embodiment, the peptide consists of the amino acid sequence shown in one of SEQ ID NO: 2 to 6 and SEQ ID NO: 37. In another preferred embodiment, the fragment comprises or consists of any sequence within the sequence defined by SEQ ID NO: 37.

[0096] The sequences set forth in SEQ ID NOs: 2-6 have been previously identified as immunodominant peptides due to their recognition by disease-associated T cells, and subsequent validation of recognition by CSF-infiltrating bulk T cells (WO2020 / 002674).

[0097] The amino acid sequence is listed below in Table 2. The gene sequence of TSTA3 (encoding GDP-L-FS) can be identified by the NCBI reference sequence: NC_000008.11 (REGION:143612618..143618048).

[0098] The following nucleotide sequences show preferred nucleotide sequences encoding the GDP-L-FS (gene name: TSTA3) of the present invention or a fragment, derivative, or splice variant thereof. Also included are coding sequences (CDS) (i.e., proteins or peptides) that can be used in the treatment of MS: RNA:XM_011517269.1, NM_003313.3, NM_001317783.1, XM_005251051.3 Proteins: XP_011515571.1, NP_003304.1, NP_001304712.1, XP_005251108.2

[0099] All sequences were searched from the respective online databases on June 22, 2018.

[0100] The following characteristics indicate that a particular peptide of a protein is immunodominant from an MS perspective: a) frequent recognition of this peptide by T cells (i.e., by approximately 10% or more of MS patients), often in the context of disease-associated HLA alleles or haplotypes (Sospedra and Martin, 2005), and b) Recognition of this peptide by disease-associated T cells, such as those that respond to the peptide at low concentrations (high avidity T cells) (Bielekova et al., 2004) and are therefore considered particularly dangerous and / or that have a pro-inflammatory phenotype and / or that are isolated from the target organ or compartment (CNS) (in the case of MS, T cells infiltrating the brain, spinal cord or CSF).

[0101] However, high avidity recognition is not a prerequisite, as low avidity myelin-specific T cells have also been shown to be pathogenic in a humanized transgenic mouse model (Quandt et al. 2012).

[0102] Thus, it is possible to test whether a protein or a fragment, derivative, or splice variant thereof is immunodominant in the context of MS. Such tests are preferably in vitro tests. T cells and / or antibodies obtained from the blood, CSF, or other body fluids of a human subject diagnosed with MS, preferably CSF-infiltrating CD4 + Particularly suitable are in vitro tests which allow the measurement of the reactivity of T cells to the protein or fragments, derivatives or splice variants to be tested. + Know how to test for T cell and / or antibody reactivity. For example, CD4 +The proliferation of T cells and / or the secretion of IFN-γ of said cells or the reactivity in ELISPOT / FLUOROSPOT assays or the reactivity to HLA-peptide tetramers can be tested. If the tested protein, or its fragment, derivative, or splice variant induces reactivity in human subjects diagnosed with MS, the tested protein, or its fragment, derivative, or splice variant can be called immunodominant if the T cell reactivity is particularly stimulation index (SI) >2 and / or IFN-γ secretion >20pg / ml. It is also possible to select 10 patients diagnosed with MS for such testing. If the reactivity is induced in at least two patients, the tested protein, or its fragment, derivative, or splice variant can be called immunodominant. Preferably, the 10 patients are diagnosed with RRMS according to the established revised McDonald criteria.

[0103] It has recently been demonstrated that T cells from MS patients exhibit increased in vitro proliferation in the absence of exogenous antigens ( Mohme et al., 2013 ; Jelcic et al., 2018 ). These “autologous” T cells are enriched in the CNS compartment of MS patients and can therefore be considered a peripheral blood source of brain / CSF-infiltrating T cells.

[0104] When data from in vitro T cell tests are not available or in addition to such tests, the immune recognition of peptides can also be predicted / predicted from peptides that are considered to bind well to the HLA-class I or class II alleles of an individual, and for CD8+ T cells and CD4+ T cells, respectively.Peptide binding prediction is well known to those skilled in the art.Peptide binding can be predicted by established prediction algorithms (NetMHCII-www.cbs.dtu.dk / services / NetMHCII / ; IEDB-www.iedb.org / ) and by analyzing HLA binding motifs (SYFPEITHI-www.syfpeithi.de / ).

[0105] The protein GDP-L-fucose synthase has previously been identified as immunodominant in MS and therefore as an autoantigen (WO2020 / 002674).

[0106] Binding to HLA alleles does not have to be particularly strong, and in fact immunodominance can occur even for peptides that bind poorly to HLA alleles (Muraro et al., 1997, J Clin Invest, 100(2):339-349).

[0107] In one embodiment of the invention, a GDP-L-FS protein, or a fragment, derivative or splice variant thereof, or a nucleotide sequence encoding a GDP-L-FS protein, or a fragment, derivative or splice variant thereof, is used in the treatment of MS in an MS patient, wherein CD27-Th1CD4+ cells are detected in a body fluid previously obtained from the MS patient, in particular in blood, preferably in peripheral blood, or in CSF.

[0108] Treatment includes, for example, tolerance induction.

[0109] In a preferred embodiment, the T cells and / or antibodies previously obtained from the body fluids of an MS patient respond to the protein GDP-L-FS, or to fragments, derivatives and / or splice variants thereof.

[0110] Therefore, the MS patients selected for treatment are stratified in such a way that only those patients who exhibit CD27-Th1CD4+ cells in a body fluid previously obtained from an MS patient, in particular in blood, preferably in peripheral blood, or in CSF, are treated. In particular, the MS patients are also responsive to the GDP-L-FS peptide.

[0111] In another embodiment of the present invention, at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence and / or at least one carrier bound to at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence is used in a method for inducing antigen-specific tolerance against an autoantigen in an MS patient, wherein CD27-Th1CD4+ cells are detected in a body fluid previously obtained from the MS patient, in particular in blood, preferably in peripheral blood or in CSF.

[0112] In a preferred embodiment, the T cells and / or antibodies previously obtained from the body fluids of an MS patient respond to the protein GDP-L-FS, or to fragments, derivatives and / or splice variants thereof.

[0113] Therefore, tolerized patients are selected according to the above criteria.

[0114] Tolerance induction is antigen-specific and induces Treg cells that render autoreactive T cells non-functional or anergic, or specifically suppress inappropriate autoimmunity against the target immunity. Induction of tolerance to target autoantigens is a very important therapeutic goal in autoimmune diseases. Tolerance induction provides an opportunity to specifically weaken pathogenic autoimmune responses in an effective manner with few side effects. Also, instead of or in addition to immunodominant peptides being fragments of proteins, tolerance induction can be achieved by applying whole proteins (Kennedy et al., 1990).

[0115] Thus, immunodominance of the protein and / or fragments allows the use of the protein, and / or its fragments, derivatives or splice variants for antigen-specific immunotherapy, such as tolerance induction.

[0116] According to the present invention, antigen-specific tolerization can be used in all the following forms of MS: At the time of first manifestation, if the differential diagnosis has been excluded, the disease is called CIS, provided that the CSF and MRI findings are consistent with the diagnosis. MRI reveals lesions in locations typical of MS (i.e., juxtacortical, periventricular, brainstem or spinal cord). RRMS can be diagnosed if certain criteria are met, which can be summarized as spatial multifocality (more than one lesion or clinical symptom / sign) and temporal multifocality (more than one event). The spatial scenario is the chance discovery of MRI lesions compatible with MS without clinical symptoms. This is called RIS and can be considered as a precursor to CIS and RRMS. More than 80% of patients will suffer from one of these, and the majority of patients will subsequently develop the so-called SPMS. At this point, relapses / exacerbations become less frequent or stop completely, and neurological impairment gradually increases between or without relapses.

[0117] A special type of MS is PPMS, which does not experience relapses, but rather begins with a steady deterioration of neurological symptoms (e.g., walking ability). PPMS affects approximately 10% of MS patients, with equal frequency in men and women. Its onset is usually later than CIS or RRMS. With regard to causes and disease pathogenesis, PPMS is considered similar to the RIS-CIS-RRMS-SPMS described above.

[0118] Typically, MS is diagnosed according to the revised McDonald criteria. These criteria are also capable of distinguishing different forms and disease activity of MS (Thompson et al., 2018, Lancet Neurol, 17(2):162-173). The MS patient of the present invention is a human who has been diagnosed with MS.

[0119] It is preferable to apply a tolerization approach early (i.e., in RIS, CIS, and early RRMS), since the immune processes at this stage are presumed to be mediated mainly by autoreactive T lymphocytes, whereas as the disease progresses, tissue damage, so-called degenerative changes, become progressively more important. However, tolerization is meaningful as long as autoreactive T cells react against the antigen used for tolerization, and may also be meaningful during SPMS and PPMS.

[0120] In a particularly preferred embodiment, GDP-L-fucose synthase protein or its splice variant, preferably GDP-L-fucose synthase protein, is used in early (i.e., RIS, CIS, and early RRMS) tolerization approach. GDP-L-fucose synthase protein has, for example, the sequence set forth in SEQ ID NO:1.

[0121] The method for inducing tolerance preferably comprises the step of applying to an MS patient in need thereof (i.e., to a human subject) at least one GDP-L-FS protein as described herein, or a fragment (peptide), derivative, and / or splice variant thereof, a nucleotide sequence encoding the protein, or any of its fragments, derivatives, or splice variants, and / or a gene sequence, or applying at least one carrier comprising at least one protein, fragment, derivative, splice variant, nucleotide sequence, and / or gene sequence as described herein.

[0122] It is particularly preferred to use the entire protein of GDP-L-fucose synthase (SEQ ID NO: 1) to induce antigen-specific tolerance. In another preferred embodiment, a fragment (peptide) of the protein is used. It is particularly preferred to use a fragment according to any of SEQ ID NOs: 2 to 6 and SEQ ID NO: 37. In another preferred embodiment, the fragment comprises or consists of any sequence within the sequence defined by SEQ ID NO: 37.

[0123] In a preferred embodiment, a GDP-L-FS protein or peptide or corresponding nucleotide or gene sequence, preferably at least one of the peptides (or corresponding nucleotide or gene sequences) GDP-L-FS51-65 (SEQ ID NO:2), GDP-L-FS136-150 (SEQ ID NO:3), GDP-L-FS161-175 (SEQ ID NO:4), GDP-L-FS246-260 (SEQ ID NO:5), GDP-L-FS296-310 (SEQ ID NO:6) and GDP-L-FS226-270 (SEQ ID NO:37), and also one or more of the following peptides (or corresponding nucleotide or gene sequences) may be used for tolerance induction: MBP13-32 (SEQ ID NO: 7) MBP83-99 (SEQ ID NO: 8) MBP111-129 (SEQ ID NO: 9) MBP146-170 (SEQ ID NO: 10) MOG1-20 (SEQ ID NO: 11) MOG35-55 (SEQ ID NO: 12) PLP139-154 (SEQ ID NO: 13)

[0124] Also, preferred embodiments that may be used for tolerance induction may comprise (instead of or in addition to one or more of the above peptides) at least one peptide (or corresponding nucleotide or gene sequence) whose sequence is within the stretch of GDP-L-FS226-270 (SEQ ID NO: 37). Preferably, this stretch within the stretch of GDP-L-FS226-270 (SEQ ID NO: 37) comprises 10 to 20 amino acids, preferably 15 amino acids.

[0125] In one embodiment, nucleotide sequence or gene sequence is applied to patient via carrier (e.g., cell).Antigen can then be expressed by carrier (e.g., cell).Transfer of RNA / DNA encoding autoantigen into carrier (e.g., cell), resulting in the coding of said autoantigen, is also considered to be similar to tumor vaccination approach using RNA encoding antigen.

[0126] At least one protein, fragment, derivative, splice variant, nucleotide sequence, and / or gene sequence may be applied by nasal, inhalation, oral, subcutaneous (sc), intracavitary (ic), intramuscular (im), intradermal (id), transdermal (td) or intravenous (iv) administration, preferably by a route of administration considered to be tolerogenic, e.g. iv, sc, id, td, oral, inhalation, nasal, or by binding to a tolerogenic carrier, preferably red blood cells (RBCs). The carrier is preferably applied systemically, in particular intravenously.

[0127] In particular, the methods may be used to induce antigen-specific tolerance to self-antigens in early MS or even in the preclinical stages of the disease.

[0128] The antigen-specific tolerance protocol provided herein can selectively target both activated and naive autoreactive T cells specific for multiple potentially encephalitogenic epitopes that perpetuate the disease.

[0129] Also, a tolerization approach can be used to prevent MS. This approach can include identifying individuals at high risk of developing MS (e.g., in families of MS patients selected as disclosed herein). For example, it is possible to tolerize children of mothers with MS or identical twins of MS patients who would be at particularly high risk of developing MS.

[0130] MS or one of its forms is diagnosed by demonstrating neurological deficits and / or MRI lesions that are compatible with spatially and temporally multiple MS. The present invention can be used to identify patients who are particularly likely to benefit from antigen-specific tolerance induction (i.e., antigen-specific tolerance approach can be personalized). By identifying patients who are particularly likely to benefit from antigen-specific tolerance induction, patients can be diagnosed in vitro with MS, particularly with a subtype of MS. This in vitro test can therefore complement clinical and imaging findings (i.e., MS diagnosis according to the prior art, particularly according to the revised McDonald criteria).

[0131] The patient subpopulation that can be identified by the present invention has potentially aggressive forms of MS. Aggressive forms can be characterized by neurodegenerative and / or neuroinflammatory processes, particularly early in the disease. Therefore, the identification of the patient subpopulation can already be possible early in the disease. In addition to the detection of CD27-Th1CD4+ cells, preferably CCR7-CD45RA-CD27-Th1CD4+ cells, and reactivity to the protein GDP-L-FS, or its fragments, derivatives and / or splice variants, in the patient's body fluids, specific characteristics can be: - Inflammation and / or neurodegeneration in the central nervous system specifically characterized by Gd-contrast enhanced T1 lesions and / or FLAIR T2 lesions, - higher expression of genes associated with Th1 cells or cytotoxicity, and / or genes encoding proinflammatory cytokines such as IL-2 and / or IFN-γ, compared to healthy controls; and / or -HLA allotype HLA-DRB3 * 02:02 or DRB3 * 03:01.

[0132] Those skilled in the art are aware of methods for detecting inflammation and / or neurodegeneration in the CNS of a patient.For example, in this regard, magnetic resonance imaging (MRI) can be used, which is a standard imaging technique for identifying demyelinating lesions.The most common MRI sequences are T1-weighted and T2-weighted scans, which can be further refined by Gd-contrast enhancement or fluid-suppressed inversion recovery (FLAIR), respectively.

[0133] Higher expression of genes associated with Th1 cells or cytotoxicity and / or genes encoding proinflammatory cytokines such as IL-2 and / or IFN-γ compared to healthy controls may, for example, be measured in body fluids, in particular blood, preferably peripheral blood, or cerebrospinal fluid (CSF), by standard techniques.

[0134] Detection of HLA allotypes is particularly important for MS stratification.

[0135] Patient subpopulations that may be identified by the present invention may also be characterized by elevated levels of biomarkers, preferably neurofilament (NF-L) and / or chitinase (YKL-40), in the CSF of MS patients (compared to healthy controls), and MS patients preferably show responsiveness to the protein GDP-L-FS, or fragments, derivatives and / or splice variants thereof.

[0136] Therefore, the biomarkers neurofilament (NF-L) and / or chitinase (YKL-40) may be used for the identification of MS patient subpopulations. Thus, in one embodiment, the use of biomarkers, particularly neurofilament (NF-L) and / or chitinase (YKL-40), is disclosed for the identification of MS patient subpopulations, where the levels of neurofilament (NF-L) and / or chitinase (YKL-40) in body fluids, preferably CSF, of MS patients are detected and compared with the levels of neurofilament (NF-L) and / or chitinase (YKL-40) in body fluids, preferably CSF, of healthy controls, where the body fluids, preferably CSF, have been previously obtained from MS patients and healthy controls.

[0137] Also, patient subpopulations that may be identified by the present invention may be characterized by elevated levels (increased frequency and / or absolute numbers) of CD4+ memory (central memory (CM) and effector memory (EM)) Th1 cells in CSF and / or paired blood, particularly peripheral blood, in patients who respond to protein GDP-L-FS, or fragments, derivatives, and / or splice variants thereof, compared to MOG(35-55) responders and / or non-responders. In particular, the CD4+EM Th1 (CD28+CD27-CCR6-CCR4-CRTh2-) population may be enriched. Therefore, CD4+ memory Th1 cells, in particular CD4+EM Th1 (CD28+CD27-CCR6-CCR4-CRTh2-) cells, can be used as a biomarker for the identification of MS patient subpopulations, where the levels of CD4+ memory Th1 cells, in particular CD4+EM Th1 (CD28+CD27-CCR6-CCR4-CRTh2-) cells are detected in the CSF and / or paired blood, in particular peripheral blood, of GDP-L-FS responders (responsive to protein GDP-L-FS, or fragments, derivatives, and / or splice variants thereof) and compared with MOG(35-55) responders and / or non-responders, where the CSF and blood have been obtained previously from MS patients. Identification of this patient subpopulation may be possible especially early in the disease.

[0138] By identifying patients or subpopulations of patients with existing and / or particularly strong proinflammatory (potentially harmful) T cell or antibody responses against the respective autoantigens, patients can therefore be diagnosed in vitro with MS, particularly MS subtypes, at an early stage of the disease.In this situation, it may be possible to tailor the tolerization treatment (e.g. the composition of peptides / proteins used for tolerization) to individual patients or subpopulations of patients, with the aim of tolerizing as specifically as possible and avoiding potential adverse effects, by pre-testing the patients with a suitable test to assess whether they belong to a specific subgroup.However, antigen-specific tolerization can also be performed in patients who have not shown a T cell response against the tolerization antigen.

[0139] In one aspect of the invention there is provided CD27-Th1CD4+ cells for use in a method of monitoring a response to a method for inducing antigen-specific tolerance as described above, wherein the CD27-Th1CD4+ cells are detected in a body fluid previously obtained from an MS patient, in particular blood, preferably peripheral blood, or CSF.

[0140] In a preferred embodiment, furthermore, the reactivity of T cells and / or antibodies previously obtained from body fluids of MS patients against the protein GDP-L-FS as defined above, or against fragments, derivatives and / or splice variants thereof, is detected.

[0141] Monitoring the response to the method for inducing antigen-specific tolerance preferably means that the success of tolerance induction can be regulated.If tolerance induction is successful, the number of CD27-Th1CD4+ cells (particularly those that are also negative for markers CCR7 and / or CD45RA) in body fluids (particularly blood, preferably peripheral blood, or CSF) decreases during the course of tolerance induction.Therefore, to monitor the response to the method for inducing antigen-specific tolerance, preferably measure the number of CD27-Th1CD4+ cells (particularly those that are also negative for markers CCR7 and / or CD45RA) in body fluids.It can be measured, for example, by flow cytometry, or by using oligonucleotide-labeled antibodies and subsequent sequencing or PCR-based methods, or by any other method that allows cells to be quantified, for example, by using antibodies and suitable detection methods.

[0142] In one embodiment, the response is monitored 4 to 12 weeks after tolerization, preferably 6 to 10 weeks after tolerization, preferably 8 weeks after tolerization. Tolerization preferably refers to the date of tolerance induction in the patient, i.e. the date of application of at least one GDP-L-FS protein, or a fragment (peptide), derivative, and / or splice variant thereof, a nucleotide sequence encoding a protein, or any of its fragments, derivatives, or splice variants, and / or a gene sequence, as described herein, to an MS patient in need of application, i.e. the date of application of at least one carrier comprising at least one protein, fragment, derivative, splice variant, nucleotide sequence, and / or gene sequence, as described herein, to an MS patient in need of application, i.e. the date of application of at least one carrier comprising at least one protein, fragment, derivative, splice variant, nucleotide sequence, and / or a ...

[0143] In one embodiment, the response to tolerance induction can be monitored for a longer period (eg, retested every 6 months) to test for maintenance of the tolerizing effect.

[0144] In one aspect of the present invention, there is provided a carrier bound to at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence as defined above for use in a method for inducing antigen-specific tolerance to an autoantigen in an MS patient, wherein CD27-Th1CD4+ cells are detected in a body fluid previously obtained from an MS patient, in particular in blood, preferably in peripheral blood, or in CSF.

[0145] The skilled person is familiar with possible carriers. For example, the carrier can be any cell, protein, lipid, glycolipid, bead, nanoparticle, virus-like particle (VLP), or molecule (such as sugar molecule), or any combination thereof, that is suitable for application in humans and to which the protein and / or fragment can be coupled, for example, by chemical coupling process, preferably by EDC. The carrier can be derived from a naturally occurring or synthetic carrier. Preferably, the cell, molecule, bead, nanoparticle, or VLP is biodegradable in vivo, or at least applicable to living humans, and degrades in vivo or is eliminated from the body to which the carrier is applied. The term cell also includes cell precursors (e.g. RBC precursors). Preferably, the carrier is a blood cell, even more preferably a red blood cell or a white blood cell. The white blood cell can be a splenocyte or a PBMC, or generally an APC.

[0146] In one embodiment, the proteins, fragments, derivatives and / or splice variants are expressed by cells, preferably blood cells, whereby the genetic information encoding the proteins, fragments, derivatives and / or splice variants is introduced into the cells before the proteins, fragments, derivatives and / or splice variants are expressed by the cells.

[0147] Any coupling agent or method for binding a protein and / or its fragment to a carrier may be used. For example, a synthetic or natural linker may be used for coupling. One example of such a linker is glycophorin A present on the surface of red blood cells (RBC). In one embodiment, chemical cross-linking is performed. In a preferred embodiment, a chemical cross-linking agent EDC is used, which catalyzes the formation of peptides between free amino and carboxyl groups. In particular, in the absence of EDC, multiple peptides can be coupled to the surface of the carrier, thereby making it possible to target multiple T cell specificities simultaneously. Preferably, more than 3, more than 5, more than 10, more than 15, or even more than 20 different peptides are coupled to the surface of the carrier. In a preferred embodiment, between 5 and 20, preferably between 5 and 15 different peptides are used. A peptide is different from another peptide if it does not consist of the same amino acid sequence. The carrier is preferably, but not necessarily, a cell. EDC can be used for coupling to any carrier as long as a free amino group is present.

[0148] In another embodiment, at least one peptide of the GDP-L-FS protein according to the invention, in particular at least one of the peptides defined by SEQ ID NO: 2 to 6 and SEQ ID NO: 37 (or any peptide present within the sequence defined by SEQ ID NO: 37), is used together with a peptide of the prior art, in particular at least one myelin peptide, in particular at least one or all of the myelin peptides defined by SEQ ID NO: 7 to 13.

[0149] In one preferred embodiment, the carrier is a blood cell, which is chemically coupled to at least one protein, fragment, derivative, and / or splice variant by a coupling agent, preferably EDC. A method for producing such chemically coupled, i.e. antigen-bound, blood cells comprises, for example, isolating blood cells from a human subject, adding at least one protein, fragment, derivative, and / or splice variant (i.e. antigen), and then adding a coupling agent, preferably EDC.

[0150] The mechanism of action of peptide-coupled cells by EDC is not fully understood but involves covalent cross-linking of peptide amino and carboxy groups to cell surface molecules, subsequent programmed cell death (apoptosis in the case of nucleated cells; eryptosis in the case of RBCs) of the peptide-coupled (i.e., antigen-coupled) cells, and subsequent tolerogenic presentation of the dying cells in vivo.

[0151] The dose of EDC used for coupling reaction can be set to maximize safety with maximum effectiveness. At high concentration, EDC can lyse cells, especially RBC. For optimal safety of RBC, a final concentration of EDC less than 15mg / ml, preferably less than 10mg / ml, even more preferably less than 5mg / ml, even more preferably about 3mg / ml can be used. Also, optimal dose can vary. Those skilled in the art know how to determine the optimal stability of RBC and the optimal dose of EDC.

[0152] The proteins, fragments, derivatives, and / or splice variants to be coupled can be added in amounts readily determined by those of skill in the art, who are aware of the means to determine the optimal amount that interacts with the optimal amount of EDC.

[0153] The incubation time can be varied to suit each specific coupling reaction (e.g., 15 min, 30 min, 45 min, 60 min, 120 min). Longer incubation times can be used to obtain higher coupling efficiency. In one embodiment, a maximum is reached after 60 min.

[0154] The incubation temperature may also vary. For example, 15-25° C. or 2-8° C. may be used. In one embodiment, the coupling efficiency is higher when the coupling reaction is carried out at 15-25° C.

[0155] Any excipient that allows the coupling reaction may be used. In one embodiment, the excipient is sterile and endotoxin-free. In a preferred embodiment, the excipient is sterile, endotoxin-free saline (NaCl 0.9%). Saline is approved for human use and provides maximum safety.

[0156] Those skilled in the art know how to determine optimal incubation times and temperatures as well as possible excipients. EXAMPLES

[0157] material and method Patient samples Paired CSF and blood samples were collected from 105 treatment-naïve MS patients, 11 control patients (CP) and 10 MOGAD patients negative for anti-AQP4 antibodies from whom only CSF was collected, and 4 healthy donors (HD) from whom only peripheral blood mononuclear cells (PBMCs) were collected (Table 1). 84 MS patients (80%) had never received treatment, whereas 21 (20%) had been previously treated but were considered treatment-naïve at the time of lumbar puncture (Table 1). Patients and controls were recruited from the NIMS-Division of Neuroimmunology and MS Research, Department of Neurology, University Hospital Zurich. MS diagnosis was based on the revised McDonald criteria (Polman et al. 2011, Ann Neurol, 69(2):292-302).

[0158] [Table 1]

[0159] Standard Protocol Approval, Registration, and Patient Consent The Ethics Committee of the Canton of Zurich approved the study procedures (EC-No. 2013-0001). Informed consent was obtained from all patients.

[0160] CSF and serum measurements _ENREF_20 Albumin index and CSF specific oligoclonal bands (OCB) were analyzed as previously reported (Puthenparampil et al., Neurol Neuroimmunol Neuroinflamm, 2021, 8(2):e951). Intrathecal Ig synthesis (Ig(loc)) was calculated according to Reiber (Reiber et al., 2009, Mult Scler, 15(12):1466-1480).

[0161] Cell culture and stimulation PBMCs were freshly isolated using Ficoll (Eurobio, Germany) density gradient centrifugation. CSF-infiltrating CD4 + T cells were expanded with PHA in one round of stimulation using a previously reported protocol aimed at reducing TCR repertoire bias (Planas et al., 2008, Sci Transl Med, 10(462):eaat4301). 4 PHA-expanded CSF infiltrate CD4 + T cells were cultured at 2 × 10 5 The cells were seeded in quadruplicate with irradiated autologous PBMCs and stimulated with peptides at a final concentration of 10 μM (Table 2) and a T cell activation kit (anti-CD3, anti-CD28, anti-CD2 beads) (Miltenyi Biotec) as a positive control.

[0162] [Table 2]

[0163] Proliferative response Proliferation was measured 72 hours after stimulation using 3H-thymidine (Hartmann Analytic, Braunschweig, Germany) as previously reported (Planas et al., 2018). Stimulation index (SI) of single wells was calculated as follows: SI = (cpm wells with peptide) / (mean cpm wells without peptide). Wells were considered positive if their SI was ≥2. Patients were considered positive for a peptide if the mean SI of 4 wells was ≥2 and at least 3 of 4 wells showed an SI ≥2.

[0164] Cytokine quantification IFN-γ release was measured using ELISA MAX™ Deluxe Set Human IFN-γ (Biolegend, San Diego, CA, USA) according to the manufacturer's instructions. Wells were considered positive if IFN-γ was ≥ 20 pg / ml, and patients were considered positive for a peptide if the mean IFN-γ of four wells was ≥ 20 pg / ml and at least three of the four wells were positive.

[0165] Cytokines in supernatants and CSF were measured using the Human T-helper Cytokine Panel LEGENDplex bead-based immunoassay (Biolegend) according to the manufacturer's instructions.

[0166] Immunophenotyping Ex vivo immunophenotyping of intrathecal and paired circulating lymphocytes was performed in a subcohort of 66 MS patients (Table 1, supra), as previously reported (Puthenparampil et al., 2021; Brodie et al., 2016, Cytometry, 89(7):629-32). _ENREF_24Absolute counts were performed using SPHERO™ AccuCount Particles (Spherotech, Inc. Lake Forest, IL, USA) added according to the manufacturer's instructions. Samples were acquired using an LSR Fortessa cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and FACSDiva (BD) and FlowJO (TreeStar Inc., Ashland, OR, USA) software was used for analysis. The gating strategy is summarized in Figure 1.

[0167] Cell subset sorting PBMCs from four MS patients and four age- and sex-matched HD patients were labeled with antibodies against CD4 (APC), CD45RA (BV711), CCR7 (BV421), CD27 (PE), and CD28 (APC-Cy7) (all BioLegend) and live-dead aqua dye (Invitrogen). Live CD4+CCR7-CD45RA-CD28+CD27+ and CD4+CCR7-CD45RA-CD28+CD27- cells were sorted using 100 μm sorting tips on a Sony SH800SFP cell sorter (4 lasers, Sony). 20,000 sorted cells from each cell population were transferred to RNase-free tubes, resuspended in Qiazol (QIAGEN, Germany), and frozen at -80°C.

[0168] RNA extraction, sequencing, and analysis RNA extraction from frozen cell pellets was performed using the PicoPure RNA isolation kit (Life Technologies) according to the manufacturer's instructions. RNA sequencing (RNAseq) was performed using an Illumina Sequencing 200M at the Zurich Center for Functional Genomics as previously described (Jelcic et al., 2018, Multiple Sclerosis Cell, 175(1):85-100). RNAseq data analysis consisted of: (i) cleaning of raw reads using Trimmomatic (version 0.36) (Bolger et al., 2014, Bioinformatics, 30(15):2114-2120); (ii) pseudoalignment of sequences against the human reference genome (build GRCh38.p13, gene annotations from GENCODE Release32) and quantification of gene expression using Kallisto (version 0.44) (Bray et al., 2016, Nat Biotechnol, 34(5):525-527); (iii) read alignment using STAR (v2.7.3) (Bray et al., 2016), and (iv) software package EdgeR (R version: 3.6.1, EdgeR version: 3.28.0) (Robinson et al. Detection of differentially expressed genes using a count-based negative binomial model implemented in (Frank et al., 2010, Bioinformatics, 26(1):139-140). Differential expression was assessed using a generalized linear model fitted to overdispersed data. Genes showing a p-value for expression change less than 0.001 were considered differentially expressed.

[0169] ELISA The following ELISA kits: NF-L (Human Diagnostics, Umea, Sweden); CXCL13 / BLC / BCA-1, granzyme A and granulysin (R&D System, MN, USA); CHI3L1 (MicroVue, Athens, OH, USA); perforin, granzyme B and granzyme H (Invitrogen-Thermo Fisher Scientific, MA, USA) were used according to the manufacturers' instructions.

[0170] HLA typing Patients were randomly assigned to HLA-class I (A * and B. * ) and HLA class II (DRB1 * , DRB3 * , DRB4 * , DRB5 * , DQA1 * , and DQB1 * ) were classified into

[0171] MOG-Antibody Detection Assay Serum and CSF samples were analyzed for MOG IgG antibodies as previously described (Reindl et al., 2020, Neurol Neuroimmunol Neuroinflamm, 7(2)). For screening, serum samples were diluted 1:20 and 1:40, and CSF samples were diluted 1:2. Positive samples were end-point titrated as recently described (Reindl et al., 2020), and MOG-IgG positivity was confirmed using an anti-human IgG (Fc) specific secondary antibody.

[0172] MRI Eight patients were scanned using a 3T Philips Ingenia and 22 patients were scanned using a 3T Siemens Skyra. The MRI protocol included a 3D pre- and post-gadolinium-enhanced gradient echo pulse sequence (MPRAGE) and a 3D fluid-suppressed inversion recovery (FLAIR) sequence.

[0173] From FLAIR, the number and total volume (ml) of all hyperintense lesions were determined by an automated algorithm based on a convolutional neural network (Kruger et al., 2020, Neuroimage Clin, 28:102445). All results were manually corrected by two experienced technical raters. Discrepancies in correction were resolved by consensus in a second reading phase. Similarly, the number of contrast-enhancing lesions was determined.

[0174] Whole brain, gray matter, and thalamic volumes (ml) were determined on pre-contrast MPRAGE images using the automated processing pipeline Biometrica MS® analysis platform (version 2.1, jung diagnostics GmbH, Hamburg, Germany) (Schippling et al., 2017, J Neurol, 264(3):520-528).

[0175] statistics Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, California, USA). Unpaired T-tests were used to compare two groups of normally distributed variables, and U-tests (Mann-Whitney) were used for non-normally distributed variables. For comparison of more than two patient groups, Kruskal-Wallis tests were used for non-normally distributed variables. Spearman r was used to test linear correlations between non-normally distributed variables. The significance level was set at p<0.05. Associations between patient specificities and seasonal distribution of LP and HLA were tested using Fisher's exact test at a significance level of 5%.

[0176] result Identification of GDP-L-FS and myelin responders CSF-infiltrating CD4 from 105 MS patients against five immunodominant GDP-L-FS ( Planas et al., 2018 ) and seven myelin peptides ( Bielekova et al., 2004 ).+ T cell proliferation and IFN-γ release are shown in Figure 2A+B. The GDP-L-FS peptide yielded the highest frequency of positive wells when proliferation was used as the readout, and the MOG(35-55) peptide yielded the highest frequency of positive wells when IFN-γ release was used (Table 3).

[0177] [Table 3]

[0178] Responses to a viral / bacterial peptide pool (CEF) (Figure 2A+B and Table 3) were also analyzed. IFN-γ secretion identified more positive wells than proliferation for the myelin and CEF peptides, whereas comparable results were obtained for the GDP-L-FS peptide (Figure 2C). Thus, the strongest correlation between proliferation and IFN-γ was found for the GDP-L-FS peptide (Figure 2D).

[0179] The frequency of positive responses to the different peptides in the patients is summarized in Table 3. In 14 patients (13.34%), the main reactivity in proliferation and / or IFN-γ release was to several GDP-L-FS peptides and classified as GDP-L-FS responders (Figure 3A+B). Of these, 6 patients (42.8%) also showed a positive response to myelin peptides. The relationship between GDP-L-FS reactivity and reactivity to myelin peptides was significant (p=<0.0001, Fisher's exact test). Four patients (3.8%) responded only to MBP peptides and were classified as MBP responders, while 11 patients (10.4%) responded only to MOG(35-55) and were classified as MOG(35-55) responders. 76 patients (72.4%) did not respond to any autoantigens and were classified as non-responders (Figure 3C+D). Responses to CEF peptides were comparable in the different patient groups, except for MOG(35-55) responders, which had a lower response (Figure 3A+B). Due to the low number of MBP responders (<5), this group was not analyzed further.

[0180] Differences in CSF-infiltrating and circulating T cells in GDP-L-FS responders Immunophenotyping of CSF-infiltrating and circulating lymphocytes _ENREF_22 was performed in a subcohort of MS patients (Table 1, supra) consisting of: GDP-L-FS responders (n=7), MOG(35-55) responders (n=7), and non-responders (n=52) (Table 4).

[0181] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8]

[0182] Effector memory (EM, CCR7) cells expressing CD28 but not CD27 (EM CD27-) - CD45RA - )CD4 + T cells were significantly more abundant in the CSF of GDP-L-FS responders (Figures 4A-B and 5A). Among these EM CD27- cells, Th1 (CCR6 - CCR4 - CRTh2 -Only cells with a functional phenotype (EM CD27-Th1) showed a significantly higher frequency (Figures 4B and 5B). Analysis of paired blood samples demonstrated that these cells were also significantly more abundant in frequency and absolute numbers in blood (Figures 4A and C and Figures 5C-F). Moreover, TEMRA (CCR7 - CD45RA + ) Differentiation state and costimulatory molecule CD28 + CD27 - and CSF infiltrates CD4 with CD28- + T cells and circulating CD4 + T cells were more abundant in GDP-L-FS responders ( Fig. 5A and C–F ).

[0183] Transcriptional signature of EM CD27-CD4+ T cells in GDP-L-FS responders EM CD27- and CD27+CD4 + T cells were sorted from peripheral blood of four GDP-L-FS responders (Figure 6A) and the transcriptomes were analyzed by RNAseq. We identified 265 differentially expressed genes (1.5-fold change, p<0.001), with 119 upregulated and 146 downregulated in EM CD27- versus EM CD27+ cells (Figure 7A). The upregulated genes included: (i) transcripts associated with cytotoxic CD8+ (Hidalgo et al., 2008, Am J Transplant, 8(3):627-636) and CD4+ (Patil et al., 2018, Sci Immunol, 3(19)) T cells, such as ADGRG1(GPR56), ADGRG5(GPR114), CCL4, CCL5, CST7, CTSW, CX3CR1, ENC1, FCRL6, FGFBP2, GNLY, GZMB, GZMH, MYO6, PRF1, PRSS23, S1PR5, SLAMF7, SPON2, TGFBR3, TRGC2, and ZEB2; (ii) transcripts associated with cytotoxic CD4+ T cells, such as ADGRG1(GPR56), ADGRG5(GPR114), CCL4, CCL5, CST7, CTSW, CX3CR1, ENC1, FCRL6, FGFBP2, GNLY, GZMB, GZMH, MYO6, PRF1, PRSS23, S1PR5, SLAMF7, SPON2, TGFBR3, TRGC2, and ZEB2; +(iii) transcription factors (TFs) important for T cell development, such as Eomes (Pearce et al., 2003, Sci, 302(5647):1041-1043) and T-bet (encoded by TBX21) (Eshima et al., 2012, Immunol Lett, 144(1-2):7-15); and (iv) other genes associated with Th1 cells, such as IFNG and GZMA. In contrast, cytokines, chemokine receptors, and TFs associated with other Th subsets (CCR4, CCR6, IL4R, IL6R, GATA3, and FoxP3) were downregulated in EM CD27- cells.

[0184] We also analyzed the same sorted EM subpopulations from four HD individuals (Figure 6A). The 265 genes that distinguished EM CD27- and CD27+ cells in GDP-L-FS responders failed to distinguish EM subpopulations in HD (Figure 7A). Differential gene expression analysis of EM CD27- cells from GDP-L-FS responders and HD identified 145 differentially expressed genes (1.5-fold change, p<0.001), of which 66 were upregulated (including 19 genes associated with cytotoxic and Th1 T cells) and 79 were downregulated (including genes associated with other T cell subsets) in GDP-L-FS responders (Figures 6B and 7A).

[0185] In EM CD27- cells from GDP-L-FS responders, there was an overall increase in transcription of genes characteristically associated with Th1 cells and cytotoxicity (TBX21, GZMA, EOMES, GNLY, GZMH, and SLAMF7) and a decrease in transcription of genes associated with other Th subsets (GATA3, CCR4, and CCR6) compared with identical cells from HD and EM CD27+ cells ( Fig. 7B ).

[0186] The genes identified in Figures 6 and 7 have the following identifiers (GENCODE database, release 32 (GRCh38.p13)): [ka]

[0187] Characterization of GDP-L-FS-specific and MOG(35-55)-specific responses GDP-L-FS specific CD4 + T cells and MOG(35-55)-specific CD4 + The functional phenotype of T cells was analyzed in the supernatants of positive wells stimulated with cognate antigen (Figure 8A+B). GDP-L-FS-specific cells released significantly higher amounts of IL-2 than MOG(35-55)-specific cells. This higher IL-2 is most likely responsible for the significantly stronger ability of GDP-L-FS-specific cells to proliferate in response to specific peptides, whereas proliferation to non-specific stimulatory beads was comparable for GDP-L-FS-specific and MOG(35-55)-specific cells (top graph in Figure 8C). IFN-γ was the main cytokine released by GDP-L-FS-specific cells, which exhibit a Th1 functional phenotype. MOG(35-55)-specific cells released, in addition to IFN-γ, cytokines associated with other Th subsets (IL-9, IL-6, and IL-10), suggesting a multifunctional phenotype (Figure 8A+B). Also, IL-10 was significantly higher in the CSF of MOG(35-55) responders, as were Th2-associated cytokines (Figure 8A+B). Several of the cytokines released by MOG(35-55)-specific cells were found to be elevated in CSF from MOGAD patients (Kaneko et al., 2018, J Neurol Neurosurg Psychiatry, 89(9):927-936), and since both patient groups share the target antigen, it was confirmed that none of the MS patients had positive anti-MOG IgG titers in serum or CSF (Table 5), and the cytokine profile in CSF from MOGAD patients was analyzed (Table 1 (supra) and Figure 9A).

[0188] [Table 5-1] [Table 5-2] [Table 5-3]

[0189] Although cytokines such as IL-9, IL-22, and IL-6 were high in both groups, the Th2-associated cytokines IL-4 and IL-10 were significantly higher in MOG(35-55) responders (Figure 9A).

[0190] Supporting the role of humoral immunity in MOG(35-55) responders, a significantly higher frequency of CSF-infiltrating naive B cells, but not circulating naive B cells, was found in these patients (bottom graph in Figure 8C). When compared to control patients (CP, Table 1, supra), the amount of intrathecal CXCL13 (Ansel et al., 2000, Nature, 406(6793):309-314) was higher in MOG(35-55) responders, and CHI3L1 (a general marker of inflammation) (Bonneh-Barkay et al., 2012, Brain Pathol, 22(4):530-546) was higher in GDP-L-FS responders (Figure 9B). Both patient groups showed significantly higher intrathecal IgG synthesis than CP (Figure 9B).

[0191] Further CSF analysis of cytotoxicity and neurodegeneration markers revealed higher levels of granzyme H, granzyme A, and neurofilament light chain (NfL) (Bergman et al., 2016, Neurol Neuroimmunol Neuroinflamm, 3(5):e271), with undetectable amounts of perforin and granzyme B, and no significant differences in granulysin, in GDP-L-FS responders (Figure 9C).

[0192] GDP-L-FS reactivity and HLA-DRB3 * Related to 02:02 / 03:01 All patients were HLA typed (Table 6).

[0193] [Table 6-1] [Table 6-2] [Table 6-3]

[0194] FIG. 10A shows the MS-associated DR15 haplotype and DRB3 * The frequency of GDP-L-FS responders, MOG(35-55) responders, and non-responders expressing the 02:02 / 03:01 locus is summarized. Based on two pre-classified MS cohorts (German cohort (n=1270) and Swiss cohort (n=367)), 40-50% of MS patients express the DR15 haplotype and 30-40% express the DRB3 haplotype. * The frequency of GDP-L-FS responders expressing the DR15 haplotype (21.4%) was lower than expected, whereas the frequency of GDP-L-FS responders expressing the DRB3 * The frequency of responders expressing the 02:02 / 03:01 locus (92.8%) was much higher. * The association between the 02:02 / 03:01 loci was significant (p=<0.0001, Fisher's exact test, Figure 10A).

[0195] Figure 10B+C shows DRB3 * GDP-L-FS peptides, myelin peptides, and CEF peptides, and CSF-infiltrated CD4 in response to stimulatory beads in patients expressing or not expressing the 02:02 / 03:01 locus + T cell responses. Responses to the GDP-L-FS peptide, both by proliferation and IFN-γ release, were greater in patients expressing DRB3 than in those expressing other HLA class II alleles. * It was significantly higher in 02:02 / 03:01 patients.

[0196] Demographic and clinical features of patients with different specificities The demographic and clinical features of GDP-L-FS responders, MOG(35-55) responders, and non-responders did not show significant differences (Table 7).

[0197] [Table 7-1] [Table 7-2]

[0198] Further characterization revealed differences in the seasonal distribution regarding the time at which LP was performed (Figure 11A+B), with a significant relationship between GDP-L-FS specificity and LP during winter / spring months (≤0.0001, Fisher's exact test).

[0199] Markers of impaired BBB permeability, disease activity, and several MRI parameters were also examined (Table 7, supra). While most of these markers were similar between GDP-L-FS and MOG(35-55) responders, semi-automated and blinded analysis of retrospectively retrieved brain MRIs from six GDP-L-FS and eight MOG(35-55) responders revealed statistically significant differences. The total number of contrast-enhanced T1 lesions and the total volume of FLAIR T2 lesions (Figure 11C) were significantly higher in GDP-L-FS responders compared to MOG(35-55) responders.

[0200] Summary of key findings This study focused on (i) CSF-infiltrating CD4 +We analyzed T cells (ii) not only for their reactivity to seven long-known immunodominant / encephalitogenic myelin peptides (Bielekova et al., 2004) but also to five recently discovered immunodominant GDP-L-FS peptides, (iii) used IFN-γ release in addition to proliferation as readout based on the implicated role of Th1 cells in MS (Bielekova et al., 2000, Nat Med, 6(10):1167-1175; Planas et al., 2018; Jelcic et al., 2018; Bielekova et al., 2004), and (iv) performed in a large cohort of 105 MS patients, thus representing the most comprehensive analysis of T cell specificity performed in MS to date. Proliferation confirmed a stronger reactivity to GDP-L-FS peptides, while IFN-γ release identified MOG(35-55) as the strongest stimulatory peptide. Fourteen GDP-L-FS responders, four MBP responders, and eleven MOG(35-55) responders were identified.

[0201] GDP-L-FS responders also frequently recognized myelin peptides.Interestingly, ex-vivo immunophenotyping demonstrated that EM CD27-Th1CD4+ were significantly more abundant in the peripheral blood as well as the CSF of GDP-L-FS responders, and that these cells in the blood expressed genes associated with Th1 and cytotoxicity (Patil et al., 2018).

[0202] Cytokine analysis indicated a role for Th1 responses in GDP-L-FS responders and Th2 responses in MOG(35-55) responders.

[0203] GDP-L-FS-specific responses were associated with DRB3 * It was associated with the 02:02 / 03:01 locus and was significantly more frequent in CSF samples obtained during winter and spring.

[0204] Finally, GDP-L-FS and MOG(35-55) responders also differed in MRI findings: it is notable that GDP-L-FS responders showed significantly higher contrast-enhanced T1 lesion counts and FLAIR T2 lesion volumes, indicating higher inflammation and more extensive demyelination in response to a more adverse immune response.

[0205] Collectively, these results reveal for the first time a link between T cell specificity and disease heterogeneity features in T cell-mediated autoimmune diseases, potentially with important implications for personalized treatment approaches aimed at antigen-specific tolerance.

[0206] Embodiment

[0207] Embodiment 1. A method for stratifying multiple sclerosis (MS) patients, comprising the steps of: - obtaining a body fluid, in particular blood, preferably peripheral blood, or cerebrospinal fluid (CSF) from an MS patient; and - detecting CD27-Th1CD4+ cells in said body fluid A method comprising:

[0208] Embodiment 2. The method further comprises: - detecting the responsiveness of T cells and / or antibodies in said body fluid to the protein GDP-L-fucose synthase (GDP-L-FS), or a fragment, derivative and / or splice variant thereof. 2. The method of embodiment 1, further comprising:

[0209] In other words, the method according to embodiment 1 is provided as embodiment 2, wherein the method comprises: - detecting the responsiveness of T cells and / or antibodies in said body fluid to GDP-L-fucose synthase (GDP-L-FS) protein, or fragments, derivatives and / or splice variants thereof. Further includes:

[0210] Particularly preferred is a method for stratifying multiple sclerosis (MS) patients, said method comprising: Obtaining peripheral blood or cerebrospinal fluid (CSF) from an MS patient; and - detecting CD27-Th1CD4+ cells in said peripheral blood or CSF, and - detecting the responsiveness of T cells and / or antibodies in said peripheral blood or CSF to the protein GDP-L-fucose synthase (GDP-L-FS), or fragments, derivatives and / or splice variants thereof. Includes.

[0211] The GDP-L-FS protein has been identified as an autoantigen. Therefore, the method according to embodiment 1 is provided as embodiment 2, wherein the method comprises: - detecting the responsiveness of T cells and / or antibodies in said body fluid to an autoantigen, wherein said autoantigen is the protein GDP-L-fucose synthase (GDP-L-FS), or a fragment, derivative and / or splice variant thereof. Further includes:

[0212] In other words, the method according to embodiment 1 is provided as embodiment 2, wherein the method comprises: - detecting the responsiveness of T cells and / or antibodies in said body fluid to an autoantigen, wherein said autoantigen is GDP-L-fucose synthase (GDP-L-FS) protein, or a fragment, derivative and / or splice variant thereof. Further includes:

[0213] Embodiment 3. The protein GDP-L-FS is a) having the amino acid sequence set forth in SEQ ID NO:1; b) has an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1; c) having an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1; d) has an amino acid sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the amino acid sequence set forth in SEQ ID NO:1, and said protein, or a fragment or splice variant thereof, binds to autologous HLA alleles, is recognized by T cells, and / or is recognized by antibodies that bind to or recognize the amino acid sequence set forth in SEQ ID NO:1 or a fragment thereof; or e) The method according to embodiment 2, wherein the TSTA3 gene is encoded by the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11 or is at least 80%, preferably at least 90%, even more preferably at least 95% identical to the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11.

[0214] Embodiment 4. The method of embodiment 2 or 3, wherein said fragment comprises 5 to 50, preferably 5 to 20, more preferably 10 to 15 amino acids, even more preferably 15 amino acids.

[0215] Embodiment 5. The fragment comprises: a) is at least 85%, preferably at least 90%, more preferably at least 95% identical to the respective corresponding amino acid sequence; b) are at least 70%, preferably at least 80%, more preferably at least 90% homologous to their respective corresponding amino acid sequences; or c) the method according to any one of embodiments 2 to 4, which is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the respective corresponding amino acid sequence, which binds to autologous HLA alleles, which is recognized by T cells, and / or which is recognized by antibodies which bind to or recognize said respective amino acid sequences.

[0216] Embodiment 6. The method of any of the preceding embodiments, wherein the fragment comprises a sequence selected from the group consisting of SEQ ID NOs:2-6 and SEQ ID NO:37, and preferably consists of a sequence selected from the group consisting of SEQ ID NOs:2-6 and SEQ ID NO:37.

[0217] Embodiment 7. A GDP-L-FS protein, or a fragment, derivative or splice variant thereof, or a nucleotide sequence encoding said GDP-L-FS protein, or a fragment, derivative or splice variant thereof, as defined in any one of embodiments 3 to 6 for use in the treatment of MS in MS patients, in which CD27-Th1CD4+ cells are detected in a body fluid previously obtained from said MS patient, in particular in blood, preferably peripheral blood, or CSF.

[0218] Embodiment 8. A GDP-L-FS protein, or a fragment, derivative or splice variant thereof, or a nucleotide sequence encoding said GDP-L-FS protein, or a fragment, derivative or splice variant thereof, for use according to embodiment 7, in which T cells and / or antibodies previously obtained from a body fluid of said MS patient respond to the protein GDP-L-FS, or a fragment, derivative and / or splice variant thereof.

[0219] In a particularly preferred embodiment, there is provided a GDP-L-FS protein, or a fragment, derivative or splice variant thereof, as defined in any one of embodiments 3 to 6, or a nucleotide sequence encoding a GDP-L-FS protein, or a fragment, derivative or splice variant thereof, for use in the treatment of MS in an MS patient, wherein CD27-Th1CD4+ cells have been detected in peripheral blood or CSF previously obtained from said MS patient, and wherein T cells and / or antibodies previously obtained from peripheral blood or CSF of said MS patient respond to said protein GDP-L-FS, or a fragment, derivative and / or splice variant thereof.

[0220] Embodiment 9. At least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence as defined in any of embodiments 3 to 6 and / or at least one carrier bound to at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence as defined in any of embodiments 3 to 6 for use in a method for inducing antigen-specific tolerance to autoantigens in MS patients, wherein CD27-Th1CD4+ cells are detected in a body fluid previously obtained from said MS patient, in particular in blood, preferably in peripheral blood or in CSF.

[0221] Embodiment 10. At least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence for use according to embodiment 9, in which T cells and / or antibodies previously obtained from said body fluid of said MS patient respond to the protein GDP-L-FS, or a fragment, derivative and / or splice variant thereof, and / or at least one carrier bound to at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence.

[0222] In a particularly preferred embodiment, there is provided at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence as defined in any of embodiments 3 to 6 for use in a method for inducing antigen-specific tolerance to an autoantigen in an MS patient, and / or at least one carrier bound to at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence as defined in any of embodiments 3 to 6, wherein CD27-Th1CD4+ cells have been detected in peripheral blood or CSF previously obtained from said MS patient, and wherein T cells and / or antibodies previously obtained from peripheral blood or CSF of said MS patient respond to said protein GDP-L-FS, or to fragments, derivatives and / or splice variants thereof.

[0223] Embodiment 11. At least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence and / or at least one carrier bound to at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence for use according to embodiment 9 or 10, wherein the at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence is applied by nasal, inhalation, oral, subcutaneous (sc), intracavity (ic), intramuscular (im), intradermal (id), transdermal (td) or intravenous (iv) administration, preferably by iv, sc, id, td, oral, inhalation or nasal administration.

[0224] Embodiment 12. CD27-Th1CD4+ cells for use in a method for monitoring a response to a method for inducing antigen-specific tolerance according to any of embodiments 9 to 11, wherein said CD27-Th1CD4+ cells are detected in a body fluid previously obtained from an MS patient, in particular in blood, preferably in peripheral blood, or in CSF.

[0225] Embodiment 13. CD27-Th1CD4+ cells for use according to embodiment 12, further comprising detecting the reactivity of T cells and / or antibodies previously obtained from said body fluid of said MS patient against protein GDP-L-FS as defined in any of embodiments 3 to 6, or a fragment, derivative and / or splice variant thereof.

[0226] Embodiment 14. CD27-Th1CD4+ cells for use according to embodiment 12 or 13, in which the number of CD27-Th1CD4+ cells in said body fluid, in particular also negative for the markers CCR7 and / or CD45RA, is reduced during the course of tolerance induction.

[0227] Embodiment 15. The method according to any one of embodiments 1 to 6, the GDP-L-FS protein, or a fragment, derivative or splice variant thereof, or a nucleotide sequence encoding a GDP-L-FS protein, or a fragment, derivative or splice variant thereof, for use according to embodiment 7 or 8, at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence for use according to any one of embodiments 9 to 11 and / or the at least one carrier bound to at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence, or the CD27-Th1CD4+ cells for use according to any one of embodiments 12 to 14, wherein the CD27-Th1CD4+ cells are additionally negative for the markers CCR7 and / or CD45RA.

[0228] Embodiment 16. The MS patient has the following characteristics: - Inflammation and / or neurodegeneration of the central nervous system specifically characterized by Gd-contrast enhanced T1 lesions and / or FLAIR T2 lesions, - higher expression of genes associated with Th1 cells or cytotoxicity and / or genes encoding proinflammatory cytokines such as IL-2 and / or IFN-γ compared to healthy controls, -HLA allotype HLA-DRB3 * 02:02 or DRB3 * 03:01 a GDP-L-FS protein, or a fragment, derivative or splice variant thereof, for use according to embodiment 7 or 8 or 15, or a nucleotide sequence encoding a GDP-L-FS protein, or a fragment, derivative or splice variant thereof, for use according to embodiment 9 or 11 or 15, and / or at least one carrier bound to at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence, for use according to embodiment 9 or 11 or 15, or a CD27-Th1CD4+ cell for use according to embodiment 12 to 14.

[0229] Embodiment 17. A method for stratifying MS patients, comprising detecting CD27-Th1CD4+ cells in a sample obtained from the patient, thereby stratifying the patient.

[0230] Embodiment 18 The method of embodiment 17, wherein the sample comprises a bodily fluid.

[0231] Embodiment 19. The method of embodiment 17 or 18, wherein the bodily fluid comprises blood, e.g., peripheral blood, or cerebrospinal fluid (CSF).

[0232] Embodiment 20. The method according to any one of embodiments 17 to 19, wherein the method comprises detecting the responsiveness of T cells and / or antibodies in the body fluid to the protein GDP-L-fucose synthase (GDP-L-FS), or a fragment, derivative, and / or splice variant thereof.

[0233] Embodiment 21. A method for treating an MS patient, comprising detecting CD27-Th1CD4+ cells in a sample obtained from the patient, and administering an MS therapy to the patient, thereby treating the patient.

[0234] Embodiment 22 The method of embodiment 21, wherein the sample comprises a bodily fluid.

[0235] Embodiment 23 The method of embodiment 21 or 22, wherein the bodily fluid comprises blood, e.g., peripheral blood, or cerebrospinal fluid (CSF).

[0236] Embodiment 24. The method according to any one of embodiments 21 to 23, wherein T cells and / or antibodies previously obtained from a sample of the patient respond to the protein GDP-L-FS, or a fragment, derivative and / or splice variant thereof.

[0237] Embodiment 25. The method of any one of embodiments 21-24, wherein the MS treatment comprises an immunodominant peptide.

[0238] Embodiment 26. The method of any one of embodiments 21 to 25, wherein the MS treatment comprises treating the patient with an antigen-specific immunotherapy, such as tolerance induction.

[0239] Embodiment 27. The method of any one of embodiments 21 to 26, wherein treating the patient comprises administering to the patient an immunodominant peptide selected from MBP, PLP, and MOG, e.g., as disclosed in EP 2205273.

[0240] Embodiment 28. The method of any one of embodiments 21 to 27, wherein treating the patient comprises administering to the patient an immunodominant protein or peptide selected from GDP-L-FS, or a fragment, derivative, or splice variant thereof, as disclosed in WO2020 / 002674, and a protein from the RASGRP family, or a fragment, derivative, or splice variant thereof.

[0241] Embodiment 29. The method of any one of embodiments 25 to 28, wherein the immunodominant peptide is, for example, chemically bound to a white blood cell or red blood cell.

[0242] Embodiment 30. Use of a GDP-L-FS protein, or a fragment, derivative or splice variant thereof, as defined in any one of embodiments 3 to 6, or a nucleotide sequence encoding said GDP-L-FS protein, or a fragment, derivative or splice variant thereof, for the manufacture of a medicament for the treatment of MS in an MS patient, wherein CD27-Th1CD4+ cells are detected in a body fluid previously obtained from said MS patient, in particular in blood, preferably in peripheral blood or in CSF.

[0243] Embodiment 31. Use according to embodiment 30, in which T cells and / or antibodies previously obtained from body fluids of an MS patient respond to said protein GDP-L-FS, or to fragments, derivatives and / or splice variants thereof.

[0244] In a particularly preferred embodiment, a GDP-L-FS protein, or a fragment, derivative or splice variant thereof as defined in any one of embodiments 3 to 6, or a nucleotide sequence encoding a GDP-L-FS protein, or a fragment, derivative or splice variant thereof, is used for the manufacture of a medicament for the treatment of MS in an MS patient, wherein CD27-Th1CD4+ cells have been detected in peripheral blood or CSF previously obtained from said MS patient, and wherein T cells and / or antibodies previously obtained from peripheral blood or CSF of said MS patient respond to said protein GDP-L-FS, or a fragment, derivative and / or splice variant thereof. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10] [Table 8-11] [Table 8-12]

Table 8-13

Claims

**Claim 1**: A method for detecting CD27-Th1 CD4+ cells as an indicator for stratifying patients with multiple sclerosis (MS), comprising: - Detecting CD27-Th1 CD4+ cells in a body fluid obtained from an MS patient, particularly blood, preferably peripheral blood, or cerebrospinal fluid (CSF). A method comprising the above step. **Claim 2**: The method further comprises: - Detecting the responsiveness of T cells and / or antibodies in the body fluid to protein GDP-L-fucose synthase (GDP-L-FS), or a fragment, derivative, and / or splice variant thereof. The method according to claim 1, further comprising the above step. **Claim 3**: The protein GDP-L-FS: a) has the amino acid sequence set forth in SEQ ID NO: 1, or b) has an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1, or c) has an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 1, or d) has an amino acid sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 1, and the protein, or a fragment or splice variant thereof, binds to a self-HLA allele, is recognized by T cells, and / or is recognized by an antibody that binds to or recognizes the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof, or e) is encoded by the TSTA3 gene, particularly the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11, or is encoded by a gene that is at least 80%, preferably at least 90%, even more preferably at least 95% identical to the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.

11. The method according to claim 2. **Claim 4**: The fragment according to claim 2 or 3 comprises 5 to 50, preferably 5 to 20, more preferably 10 to 15 amino acids, even more preferably 15 amino acids. The method according to claim 2 or 3. **Claim 5**: The fragment: a) is at least 85%, preferably at least 90%, more preferably at least 95% identical to each corresponding amino acid sequence, or b) is at least 70%, preferably at least 80%, more preferably at least 90% homologous to each corresponding amino acid sequence, or c) is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to each corresponding amino acid sequence, binds to a self-HLA allele, is recognized by T cells, and / or is recognized by an antibody that binds to or recognizes said each amino acid sequence, the method according to claim 2 or 3.

6. The method according to claim 1, wherein the fragment comprises a sequence selected from the group consisting of SEQ ID NOs: 2-6 and SEQ ID NO: 37, preferably consists of a sequence selected from the group consisting of SEQ ID NOs: 2-6 and SEQ ID NO:

37.

7. A composition for use in the treatment of MS in an MS patient, comprising the GDP-L-FS protein as defined in claim 3, or a fragment, derivative, or splice variant thereof, or a nucleotide sequence encoding the GDP-L-FS protein as defined in claim 3, or a fragment, derivative, or splice variant thereof, wherein CD27-Th1 CD4+ cells are present in a body fluid, particularly blood, preferably peripheral blood, or CSF, obtained in advance from said MS patient.

8. The composition according to claim 7, wherein the T cells and / or antibodies obtained in advance from the body fluid of said MS patient respond to the protein GDP-L-FS, or a fragment, derivative, and / or splice variant thereof. A composition for use in a method for inducing antigen-specific tolerance to self-antigens in MS patients, comprising at least one GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence, and / or gene sequence as defined in claim 3, and / or at least one carrier bound to the GDP-L-FS protein, fragment, derivative, splice variant, nucleotide sequence, and / or gene sequence as defined in claim 3, wherein CD27-Th1 CD4+ cells are detected in a body fluid, particularly blood, preferably peripheral blood, or CSF, previously obtained from said MS patient.

10. The composition according to claim 9, wherein T cells and / or antibodies previously obtained from said body fluid of said MS patient respond to the protein GDP-L-FS, or a fragment, derivative, and / or splice variant thereof.

11. The composition according to claim 9 or 10, characterized in that the composition is applied by nasal, inhalation, oral, subcutaneous (s.c.), intracoelomic (i.c), intramuscular (i.m.), intradermal (i.d.), transdermal (t.d.), or intravenous (i.v.) administration, preferably i.v., s.c., i.d., t.d., oral, inhalation, or nasal administration.

12. A composition comprising CD27-Th1 CD4+ cells for use in a method of monitoring the response to a method for inducing antigen-specific tolerance as claimed in claim 9, wherein said CD27-Th1 CD4+ cells are detected in a body fluid, particularly blood, preferably peripheral blood, or CSF, previously obtained from an MS patient.

13. The composition according to claim 12, further characterized in that the responsiveness of T cells and / or antibodies previously obtained from said body fluid of said MS patient to the protein GDP-L-FS, or a fragment, derivative, and / or splice variant thereof, as defined in claim 3 is detected.

14. The method according to any one of claims 1 to 3, the composition according to claim 7 or 8, the composition according to claim 9 or 10, or the composition according to claim 12, wherein said CD27-Th1 CD4+ cells are further negative for the markers CCR7 and / or CD45RA.

15. Said MS patient has the following characteristics: - Inflammation and / or neurodegeneration of the central nervous system, particularly characterized by -Gd contrast-enhanced T1 lesions and / or FLAIR T2 lesions, - Higher expression of genes related to Th1 cells or cytotoxicity, and / or genes encoding pro-inflammatory cytokines such as IL-2 and / or IFN-γ, compared to healthy controls, - HLA allele HLA-DRB3 ＊ 02:02 or DRB3 ＊ 03:01 The method according to any one of claims 1 to 3, the composition according to claim 7 or 8, the composition according to claim 9 or 10, or the composition according to claim 12, having one or more of the above.

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