EP4792132A1 - Prognosis method of multiple sclerosis - Google Patents

Prognosis method of multiple sclerosis

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Publication number
EP4792132A1
EP4792132A1 EP24786502.5A EP24786502A EP4792132A1 EP 4792132 A1 EP4792132 A1 EP 4792132A1 EP 24786502 A EP24786502 A EP 24786502A EP 4792132 A1 EP4792132 A1 EP 4792132A1
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Prior art keywords
cells
cell
multiple sclerosis
aggressive
cd45ra
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French (fr)
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David-Axel LAPLAUD
Emilie DUGAST
Sita SHAH
Alexandra Garcia
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Institut National de la Santé et de la Recherche Médicale
Centre Hospitalier Universitaire de Nantes
Nantes Université
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Universite de Nantes
Institut National de la Santé et de la Recherche Médicale
Centre Hospitalier Universitaire de Nantes
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Publication of EP4792132A1 publication Critical patent/EP4792132A1/en
Pending legal-status Critical Current

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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
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    • G01—MEASURING; TESTING
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70517—CD8
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70589—CD45
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

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Abstract

The invention relates to an in vitro prognosis method of an aggressive multiple sclerosis in a patient afflicted by a multiple sclerosis, the method comprising: - quantifying in a biological sample of the patient the amount of a cell called NK-like cell having a phenotype CD3+, CD8+, CD45RA-, Vα7.2-, CCR7-, and CD94+; - comparing the quantified amount of NK-like cells with a reference, and - concluding that - if the quantified amount of NK-like cells is lower than or equal to the reference, then the patient is not liable to develop an aggressive multiple sclerosis, and - if the quantified amount of NK-like cells higher than the reference, the patient will probably develop an aggressive multiple sclerosis within 24 months or more.

Description

    Prognosis method of multiple sclerosis
  • Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system (CNS) and is a major cause of non-traumatic disability in young adults. Although, the precise mechanisms behind its pathophysiology are still under study, immune cells infiltrate the CNS and play a role in driving neuroinflammation, especially T cells.
  • Historically, CD4+ T cells have been extensively studied in MS with the presence of IFNγ-producing TH1 cells and IL-17-producing TH17 cells in MS lesions, as well as their role in development of the experimental autoimmune encephalomyelitis (EAE) model. However, CD8+ T cells have become of increasing interest as these cells are predominant in MS lesions compared to CD4+ T cells and present clonal expansions in cerebrospinal fluid and CNS lesions, suggesting recognition of a local antigen. Several studies reveal that infiltrating CD8+ T cells display a memory phenotype, acquire a resident memory phenotype and produce pro-inflammatory cytokines such as IFNγ, IL-17 and TNFα, or cytotoxic molecules such as granzyme B and perforin. Furthermore, correlation between CD8+ T cell infiltration in the CNS and acute axonal injury in the CNS of patients has been reported. These results suggest a pathogenic role for CD8+ T cell populations in the CNS and that some of these T cells were also found in the periphery.
  • The evolution of the disease varies considerably among patients, due to its heterogeneous nature, with cases varying from a benign condition to a rapidly incapacitating disease. Recently, more and more observational studies have shown a long-term superiority of high efficacy treatments (HET) compared to moderate efficacy treatments (MET) when given early.
  • However, it is not known at the time of the first demyelinating event whether the patient will need MET or HET. As the time needed to decide which treatment to administer accumulates, so does the risk of tissue damage in the brain. Therefore, it would be of high interest for clinicians to be able to discriminate at disease onset which patient is at risk of severe disease and would benefit early of HET. Definition of aggressive or severe disease is based on clinical, radiological and demographical characteristics. As such, the definition of a severe disease could be summarized by the need for HET as a proxy. A better comprehension of disease mechanisms and diverse presentation among patients is therefore crucial to ensure optimal treatment.
  • Studies investigating the immunopathology of MS have largely focused on adaptive T and B lymphocytes. However, in recent years there has been an increased interest in the contribution of innate immune cells, amongst which the natural killer (NK) cells. In particular, Jing Li et al (Jing Li et al. 2022, Science, 10.1126/science.abi9591) have identified TCD8 cells having NK-like phenotype.
  • However, despite the fact that these cells are present in some auto-immune sample, and in particular in MS, no correlation has been made regarding the likelihood of relapse or of development of severe form.
  • Therefore, there is a need to provide a new and efficient method to predict the development of MS, and in particular to identify if the patients will develop severe forms of MS.
  • The purpose of the invention is to obviate the lack of prior art.
  • One aim of the invention is to provide a method for predicting the relapse in patients which is more efficient and allows the practionner to provide the most accurate treatment, by anticipating the disease evolution.
  • Another aim of the invention is to provide a new cell subset which allows to carry out the above method.
  • Still another aim of the invention is to provide a kit useful for predicting evolution of the disease. The invention relates to a prognosis method, possibly in vitro, of aggressive multiple sclerosis in a patient afflicted by multiple sclerosis, the method comprising:
  • - quantifying in a biological sample of the patient the amount of cells called NK-like cells having a phenotype CD3+, CD8+, CD45RA-, Vα7.2-, CCR7-, and CD94+, preferably cells having a phenotype CD3+, CD8+, CD4-, CD45RA-, CCR7-, CD94+, CD27-, CD62L-, CD127- and CD56-; in order to obtain quantified amount of NK-like cells,
  • - comparing the quantified amount of NK-like cells with the control amount of a NK-like cell in a control biological sample, and
  • - concluding that
  • - if the quantified amount of NK-like cells is lower than or equal to the control amount, then the patient is not liable to develop an aggressive multiple sclerosis, and
  • - if the quantified amount of NK-like cells is higher than 0.21% of total CD8 T cells, the method gives a sensitivity of 80% and a specificity of 38% to detect an aggressive multiple sclerosis within 24 months or more.
  • More advantageously, the invention relates to the method as defined above the method comprising:
  • - quantifying in a biological sample of the patient the amount of cells called NK-like cells having a phenotype CD3+, CD8+, CD45RA-, Vα7.2-, CCR7-, and CD94+, preferably cells having a phenotype CD3+, CD8+, CD4-, CD45RA-, CCR7-, CD94+, CD27-, CD62L-, CD127- and CD56-; in order to obtain quantified amount of NK-like cells,
  • - comparing the quantified amount of NK-like cells with the control amount of a NK-like cell in a control biological sample, and
  • - concluding that
  • - if the quantified amount of NK-like cells is lower than or equal to the control amount, then the patient is not liable to develop an aggressive multiple sclerosis, and
  • - if the quantified amount of NK-like cells is higher than 0.51% of total CD8 T cells, the method gives a sensitivity of 34% and a specificity of 80% to detect an aggressive multiple sclerosis within 24 months or more.
  • In the invention, sensibility relative to detection corresponds to the percentage of patients that will develop an aggressive multiple sclerosis within two years or more.
  • Sensibility can also be associated to a specificity, which provide information about false positive detection.
  • In the invention patients with the ‘aggressive’ form of multiple sclerosis accrue disability at an accelerated rate, typically reaching Expanded Disability Status Score (EDSS) ≥ 6 within 10 years of symptom onset. More precisely, patients are classified as having aggressive multiple sclerosis if they met one of the three of the following criteria: (i) EDSS ≥6 reached within 10 years of symptom onset; (ii) EDSS ≥6 confirmed and sustained over ≥6 months; and (iii) EDSS ≥6 was sustained until the end of follow-up (≥10 years).
  • Another definition of “aggressive form of MS” can be a MS form that need, for treating the patient within 2 years after disease onset, the use of a highly efficacy therapy including finglimod or other S1P modulators, natalizumab or anti-CD20 antibody.The invention is based on the surprising observation made by the inventors that a subpopulation of memory T cells, harboring natural killer (NK) cells properties, are significantly more represented in patients that will develop a relapse of multiple sclerosis (MS), or that will develop severe forms of MS.
  • These specific cells are CD3 positive (CD3+) and CD8 positive (CD8+), which means that the CD3 (cluster of differentiation 3) and CD8 (cluster of differentiation 8) transmembrane glycoproteins are expressed at the cell surface of the cell. CD8 is a co-receptor for the T-cell receptor (TCR). Along with the TCR, the CD8 co-receptor plays a role in T cell signaling and aiding with cytotoxic T cell-antigen interactions. This is therefore a signature of a T cell phenotype.
  • The above cells are also CD94 positives, which means that these cells express at their cell surface the receptor CD94 (cluster of differentiation 94, also called killer cell lectin-like receptor subfamily D, member 1 (KLRD1)). This marker is specific of NK cells, and present at low level in some activated CD8 T cells subsets.
  • Moreover, the cells do not express CCR7 and are negative (CCR7-). However, CCR7, C-C chemokine receptor type 7, has been documented to be expressed at the cell surface of naive and central memory CD8 T cells, controlling the migration of these effective cells.
  • Therefore, the cells newly identified by the inventors harbors surprisingly both antagonistic phenotypes : they are memory TCD8 cells with NK properties..
  • The inventors, by using different cohort of patients, and by compiling the patient data during a long period of time, identify that the above mentioned cells can be used for prognosing or predicting the apparition in the future of severe form of MS.
  • For this purpose, it is possible, by quantifying the amount of the NK-like cells, to give a prediction over 2 years of the MS outcomes.
  • The amount of the NK-like cells can be easily determined by flow cytometry analysis, by a minima using antibodies specifically recognizing CD3 antigen CD8 antigen, CD45RA antigen, CD94 antigen, Vα7.2 antigen and CCR7 antigen. The skilled person, having common knowledge within the field of cytometry analysis and cell sorting, could easily carry out such a detection. The Example illustrates one way to identify these cells.
  • When the amount of NK-like cells has been quantified in a biological sample of a patient afflicted by MS, i.e. in a patient that has been previously diagnosed as having a MS, this amount should be compared to a reference, i.e. a reference amount of NK-like cells, or a control amount of NK-like cells.
  • This reference amount can be obtained in many different samples:
  • - in a biological sample originating from a healthy person, i.e. a person who has not developed any specific disease, and in particular that does not present any symptoms, in particular early symptoms of MS; or
  • - in a biological of an individual that has been diagnosed as positive for MS, but that has never developed any severe form of the disease.
  • It is not recommended to use as control sample a biological sample from the patient obtained at the diagnosis, because it could be, at an early stage, representative of a future development of a severe form of MS. The risk of false negative result will be therefore high.
  • With both data, i.e. the measured amount of NK-like cells in a biological sample of a patient for who it is to be determined if he will develop a severe form of the disease, and the control amount, it is easy to provide a prediction over 2 years:
  • - if the measured amount is equal to the control amount, then the patient will not develop a severe form of MS within 2 years,
  • - if the measured amount is lower than the control amount, here again then the patient will not develop a severe form of MS within 2 years, but
  • - If the quantified amount of NK-like cells is higher than 0.21% of total CD8 T cells, the method gives a sensitivity of 80% and a specificity of 38% to detect an aggressive multiple sclerosis within 24 months or more, more advantageously if the quantified amount of NK-like cells is higher than 0.51% of total CD8 T cells, the method gives a sensitivity of 34% and a specificity of 80% to detect an aggressive multiple sclerosis within 24 months or more.
  • Advantageously, the invention relates to the method as defined above, wherein the NK-like cell having a phenotype CD3+, CD8+ , CD4-, CD45RA-, Vα7.2-, CCR7- , CD127low, CD94+ , CD27-, CD62L- and CD56-.
  • These cells are positives for the markers CD3+, CD8+ and CD94+ , but does not express CD4-, CD45RA-, Vα7.2-, CCR7- , CD127-, CD27-, CD62L-, and CD56.
  • CD3 (cluster of differentiation 3) is a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cell (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). This protein is present since the cells are memory TCD8 cells.
  • Naive T lymphocytes are typically positive for CD45RA, which is protein tyrosine phosphatase (PTP) also called leukocyte common antigen (LCA).
  • CD4 (cluster of differentiation 4) is a co-receptor for the T-cell receptor (TCR), and is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells.
  • CD127 (cluster of differentiation 127) corresponds to the interleukine-7 receptor, protein. The IL7R is expressed on naïve T cells.
  • CD27 (cluster of differentiation 27) is expressed on both naïve and activated effector T cells as well as NK cells and activated B cells.
  • CD62L (cluster of differentiation 62L) is a member of the lectine family.
  • CD56 is also called Neural cell adhesion molecule (NCAM). The expression of CD56 is mostly associated with, but not limited to, natural killer cells. CD56 has been detected on other lymphoid cells, including gamma delta (γδ) Τ cells and activated CD8+ T cells, as well as on dendritic cells.
  • Advantageously, the invention relates to the method as defined above, wherein said NK-like cells, upon IL-15 stimulation express some KIR at low level and CD56 markers.
  • Killer-cell immunoglobulin-like receptors (KIRs), are a family of type I transmembrane glycoproteins expressed on the plasma membrane of natural killer (NK) cells and a minority of T cells.
  • CD56, as well as KIR are expressed upon stimulation by IL-15. IL-15 is an inflammatory cytokine secreted by mononuclear phagocytes. This cytokine induces the proliferation of natural killer cells.
  • This confirms that the NK-like cells identified in the present invention have NK cells properties, but are not formally standard NK cells, since they express TCD8 markers.
  • Advantageously, the invention relates to the method as defined above, wherein aggressive multiple sclerosis is multiple sclerosis originating from multiple sclerosis having spinal cord lesions at onset and multiple sclerosis with a high Expanded Disability Status Scale equal to or higher than 3 and pyramidal signs at onset.
  • The invention also relates to an in vitro prognosis method of relapse of multiple sclerosis in a patient afflicted by a multiple sclerosis, the method comprising:
  • - quantifying in a biological sample of the patient the amount of a cell called NK-like cell having a phenotype CD3+, CD8+, CD45RA-, Vα7.2-, CCR7-, and CD94+ in order to obtain quantified amount of NK-like cells,
  • - comparing the quantified amount of NK-like cells with the control amount of a NK-like cell in a control biological sample, and
  • - concluding that
  • - if the quantified amount of NK-like cells is lower than or equal to the control amount, then the patient is not liable to develop a relapse of multiple sclerosis within 24 months or more, and
  • - if the quantified amount of NK-like cells is higher compared to the control amount, then the patient will be liable to develop a relapse multiple sclerosis within 12 months or more.
  • The above definitions apply mutatis mutandis.
  • Advantageously, the invention relates to the method as defined above, wherein the NK-like cell having a phenotype CD3+, CD8+, CD4-, CD45RA-, Vα7.2-, CCR7-, CD94+, CD27-, CD62L-, CD127- and CD56-.
  • Advantageously, the invention relates to the method as defined above, wherein said NK-like cell, upon IL-15 stimulation expresses KIR and CD56 markers.
  • The invention relates to an isolated and purified NK-like cell having a phenotype CD3+, CD8+, CD4-, CD45RA-, Vα7.2-, CCR7-, CD127-, CD94+, CD27-, CD62L- and CD56-.
  • As mentioned above, these cells, when isolated from the patients, are novel. They have never been identified, characterized and purified earlier.
  • Purification of these cells can be easily carried out by cell sorting by using specific antibodies, as mentioned above and in the example. The skilled person is able to determine the best way to purify these cells.
  • The invention also relates to the isolated and purified NK-like cell having a phenotype CD3+, CD8+, CD4-, CD45RA-, Vα7.2-, CCR7-, CD127-, CD94+, CD27-, CD62L- and CD56-, for its use as medicament.
  • the NK-like according to the invention can be used as a drug or medicine, in order to treat pathologies. Depending on the pathology, the practitioner would easily determine the amount of cells to use for a treatment.
  • The cells as defined above can be used per se, or combine with different ingredient(s) in order to facilitate their action. It is obviously taken into account of the immunological background of the patient receiving the cell, in order to avoid a “graft” rejection.
  • It is also disclosed here a method for treating a pathology comprising the administration to a patient in a need thereof of an effective amount of an isolated and purified NK-like cell having a phenotype CD3+, CD8+, CD4-, CD45RA-, Vα7.2-, CCR7-, CD127-, CD94+, CD27-, CD62L- and CD56-.
  • The invention also relates to the use of a NK-like cell having a phenotype CD3+, CD8+, CD45RA-, Vα7.2-, CCR7-, and CD94+, for carrying out a method for prognosing the likelihood to develop a relapse of multiple sclerosis, or to develop an aggressive multiple sclerosis.
  • Advantageously, the invention relates to the use as defined above, wherein the NK-like cell having a phenotype CD3+, CD8+, CD4-, CD45RA-, Vα7.2-, CCR7-, CD94+, CD27-, CD62L-, CD127- and CD56-.
  • Advantageously, the invention relates to the use as defined above, wherein said NK-like cell, upon IL-15 stimulation expresses KIR and CD56 markers.
  • The invention also relates to a kit comprising, consisting essentially of, or consisting of means for detecting, at the cell surface of a cell, the following markers: CD3, CD8, CD45RA, Vα7.2, CCR7, and CD94, preferably the markers CD3, CD8, CD4, CD45RA, CCR7, CD94, CD27, CD62L, CD127 and CD56.
  • Means for detecting the cells are preferably antibodies specific to each of the above markers. Preferably the kit as defined above comprises at least a specific antibody directed against CD8 protein and one antibody directed against CD94.
  • It could be advantageous that he means for detecting the above mentioned cells be a bispecific antibody. Bispecific antibodies (BsAbs) are antibodies with two binding sites directed at two different antigens or two different epitopes on the same antigen. Here, the bispecific antibody could simultaneously recognize both C94 and CD8. This means that one CDR of the antibody recognize CD8, and the other CDR recognize CD94.
  • Advantageously, the invention relates to the kit as defined above, consisting of:
  • - means for detecting, at the membrane of a cell, the following markers: CD3, CD8, CD45RA, Vα7.2, CCR7, and CD94, possibly CD3, CD8, CD45RA, Vα7.2, CCR7, and CD94, KIR and CD56 and,
  • - a biological sample comprising cells of an individua known to not having developed a relapse of multiple sclerosis or an aggressive multiple sclerosis.
  • The kit according to the invention can comprises also a control sample, that could help to carry out the methods as defined above.
  • Advantageously, the kit as defined above can also contain additionally a positive control, i.e a sample corresponding to a sample originating from a patient having developed a relapse of MS, or having developed a severe form of MS.
  • The invention relates to a compound inhibiting the cytotoxic activity of NK-like cell having a phenotype CD3+, CD8+, CD4-, CD45RA-, Vα7.2-, CCR7-, CD94+, CD27-, CD62L, CD127- and CD56-, as a drug.
  • The compound can be any molecule that can inhibit, or kill the above mentioned cells. This compound can be a chemical compound or an antibody.
  • In case of an antibody, the compound can be a bispecific antibody anti-CD8/anti-CD94, possibly associated with either a toxin or a radioisotope.
  • Since the bispecific antibody specifically identify the NK-like cells as disclosed in the present invention, the toxin or the radioisotope are specifically addressed to the target cell, and the cell is then killed.
  • The invention also relates to the compound as defined above, for its us for treating relapse of multiple sclerosis or a severe multiple sclerosis.
  • The invention will be better explained in view of the following figures and the Example.
    • Brief description of the drawings
  • - Single-cell transcriptomics reveals blood specific differences between MS patients and HV.
  • Study design (see Methods).
  • On the left panel, UMAP plot representing clusters of peripheral mononuclear cells (PBMC) identified in merged single-cell transcriptomes of blood (92396 cells) from HV (n=11), CIS patient evolving towards an aggressive MS (n=11) or a non-aggressive MS (n=11). Cluster names were assigned manually. On the right panel UMAP highlighting the expression level of CD3E (for T cells), MS4A1(for B cells) and CD14 (for monocytes) expression used to define cluster names.
  • UMAP plot representing 15 color-coded clusters of T cells identified in merged single-cell transcriptomes of blood (57827 cells) from HV (n=11), CIS patient evolving towards an aggressive MS (n=11) or a non-aggressive MS (n=11). Cluster names were assigned manually.
  • Pie chart representation of T cell cluster distribution among MS patients (outer ring) and HV (inner circle). CD8+ T cell clusters are shown on the left. CD4+ T cell clusters are shown on the right. Chi² statistical tests with a Z post test were applied. ****p<0.0001.
  • - A subset of CD8+ effector memory T cells with cytotoxic traits is increased in blood of aggressive MS patients (Agr MS) compared to non-aggressive MS (nAgr MS) patients at onset.
  • Pie chart representation of T cell cluster distribution among aggressive MS patients (outer ring) and non-aggressive MS patients (inner circle). CD8+ T cell clusters are shown on the left. CD4+ T cell clusters are shown on the right. Chi² statistical tests with a Z post test were applied. ****p<0.0001.
  • Heatmap representation of gene expression by all T cell clusters
  • Volcano plot showing differential gene expression for NK-like CD8+ T cells between Aggressive MS and non-Aggressive MS patients
  • Gene set enrichment analysis of positively differentially expressed genes between CD8em1 cells and other clusters. Wilcoxon test
  • - Flow cytometry assay confirmed NK-like CD8+ T cell increase in aggressive MS.
  • Gating strategy for NK-like CD8 T cells, with marker chosen from single-cell analysis. FACS Celesta.
  • Proportions of effector memory CD4+ T cells and naïve CD8+ T cells were quantified among CD4+ T cells and CD8+ cells respectively found in blood of HV controls (n=30) and MS patients (n=60).
  • Proportions of NK-like CD8+ T cells and CD8+ CD56+ T cells within CD8+ T cells were quantified among non-Aggressive MS patients (n=29) and Aggressive MS patients (n=31). Mann-Whitney test, *p < 0.05, **p < 0.01.
  • - NK-like CD8+ T cells discriminate aggressive MS patients from non-aggressive MS patients at onset
  • Lesion burden was recorded among non-Aggressive MS patients (n=29) and Aggressive MS patient (n=31). Chi² test
  • Receiver operating characteristic curve (ROC) for prediction of aggressive MS based on the frequency of NK-like CD8+ T cells, lesion burden and presence of optic neuritis at onset
  • Proportions of NK-like CD8+ T cells within CD8+ T cells were quantified for patients discriminated by the presence of gadolinium enhancing lesions, spinal cord lesions, or a combination of EDSS ≥ 3 and motor signs. Mann-Whitney test
  • Lesion aggravation at 1 year was recorded among non-Aggressive MS (n=28) and Aggressive MS (n=29) patients. Lesion aggravation within 2 years was recorded among non-Aggressive MS (n=28) and Aggressive MS (n=28) patients. EDA (Evidence of Disease Activity) or NEDA (Non Evidence of Disease Activity) at 1 year was recorded among non-Aggressive MS (n=23) and Aggressive MS (n=30) patients. Chi² test
  • ROC for prediction of aggressive MS based on the frequency of NK-like CD8+ T cells at onset, lesion aggravation at 1 year, EDA at 1 year, and a combination of all three factors
  • Serum levels of GFAP from patients with non-Aggressive MS (n=27) or Aggressive MS (n=25). Mann-Whitney test
  • Serum levels of Nfl from patients with non-Aggressive MS (n=29) or Aggressive MS (n=25). Mann-Whitney test
  • ROC for prediction of aggressive MS based on the frequency of NK-like CD8+ T cells, sGFAP, sNfl and combination of all three factors at onset
  • On the left: pie chart representation of CD8+ T cell cluster distribution among MS patients during relapse (outer ring) and MS patients at distance from relapse (inner circle). Chi² statistical tests with a Z post test were applied. On the right Heatmap representation of gene expression by all CD8+T cell clusters
  • Correlation between frequency of NK-like CD8+ T cells and time post relapse.
    • Example
  • The characterized peripheral T cell populations at onset in 11 patients evolving towards an aggressive multiple sclerosis (MS), compared to 11 patients evolving towards a non-aggressive MS and 11 healthy volunteers using single-cell transcriptomics. The inventors found an increase in cytotoxic effector memory CD8+ T cells proportion expressing NK markers in patients who later developed aggressive MS compared to non-aggressive MS. The inventors then performed flow cytometry to confirm these results at the protein level on a multicentric larger cohort of 62 patients. The inventors’ data suggests that a specific population of effector memory CD8+ cells, expressing NK cell markers, homing and cytotoxicity markers, is associated to disease severity and their frequency at disease onset may be used to anticipate aggressive MS. This cell subset was also associated to the relapse episode during remitting-relapsing MS as when relapse occurs, the inventor observed an increase frequency of the NK-like CD8+ T cells.
    • Materials and Methods
  • Study design
  • This study aimed to identify peripheral blood cell populations and/or markers discriminating CIS patients who will later present an aggressive form of MS from patients who will present a non-aggressive form of MS. Disease severity was evaluated according to first line treatment response at two years. Patients from Group 1 with aggressive MS were those who necessitated administration of a HET (either due to poor response to a first line platform treatment or prescribed as their first treatment) in the 2 years following the first demyelinating event. Patients from Group 2 with non-aggressive MS were those who responded well to platform therapies or did not require any treatment in the 2 years following the clinically isolated syndrome (CIS) stage.
  • The following molecules were considered as high efficacy treatments: anti-CD20, natalizumab, fingolimod. MET were composed of interferons, glatiramer acetate, teriflunomide and dimethyl-fumarate.
  • Known factors contributing to disease severity were also recorded, such as the presence of spinal cord lesions, gadolinium enhancing lesions, pyramidal signs and a high expanded disability status scale (EDSS).
  • 2 other cohorts were used to study the cell population regarding the relapse episode of RR-MS patients. The first one is a cohort of 11 RR-MS where the blood sampling was performed during relapse (n=5) or at distance from a relapse (n=7), this cohort was used for single-cell high throughput qPCR. The second cohort include 17 MS patients that have a relapse in the last 4 months and this cohort was used for flow cytometry analysis.
  • Samples
  • Blood samples from CIS patients were obtained from the multiple sclerosis centre (Centre de Ressources et de compétences sur la sclérose en plaques CRC-SEP; Pays de La Loire) of the Nantes University Hospital (ABM PFS13-003 “Collection sclérose en plaques”) and from the OFSEP biocollection (Observatoire Français de la Sclérose en Plaques, french registry of MS). MS patients were diagnosed according to the McDonald 2010 criteria and provided informed consent to participate in this study in compliance with the Declaration of Helsinki. PBMCs (peripheral blood mononuclear cell) were isolated from the blood samples using a standard Ficoll gradient according to the manufacturer’s instructions (Eurobio) and frozen until used. PBMC samples from 12 CIS patients in Group 1 with aggressive MS after 2 years, 12 CIS patients in Group 2 with non-aggressive MS after 2 years and 12 healthy volunteers were included in the study cohort. Individuals from all groups were matched in age, sex, and date of collection. Matched sample trios were pooled and processed together during the 3’ scRNAseq analysis.
  • In the validation cohort PBMC samples from 31 CIS patients in Group 1 with aggressive MS after 2 years, 31 CIS patients in Group 2 with non-aggressive MS after 2 years were provided by the OFSEP biocollection and came from 7 MS centers in France. Additionally, 30 healthy volunteers sampled in the Nantes MS center were also included. All the samples were used as a validation cohort for flow cytometry analysis. All patients were matched for sex and age at time of sampling.
  • The clinical and demographical characteristics of the three groups are summarized in the following table:
  • CIS patients (Group 2)
    (now non aggressive MS)    		 CIS patients (Group 1)
    (now aggressive MS)    		 Healthy volunteers
    Study cohort Number 12 12 12
    Age* 39.3 +/- 8.4 37.7 +/- 6.3 37.7 +/- 8.5
    Sex (M/F) 4/8 4/8 4/8
    Validation cohort Number 29 31 30
    Age* 38.68 +/- 8.5 38.9 +/- 11.1 39.2 +/- 10.8
    Sex (M/F) 11/18
  • Table 1 - Clinical and demographical patient and control characteristics - * age in years, mean +/- standard deviation (SD).
  • Generation of scRNAseq dataset
  • PBMCs from the study cohort were thawed and stained according to the CITEseq (cellular indexing of the transcriptome and epitopes by sequencing) method with barcoded antibodies targeting surface markers CCR7, CD45RA, CD56 and TCR Vα7.2 (TotalSeq™, Biolegend), and HTO (hashtag oligonucleotide) antibodies specific to each individual per pool (TotalSeq™, Biolegend).
  • Single-cell suspensions were loaded on a Chromium Chip G (10X Genomics) using the Chromium Next GEM Single Cell 3’ Library and Gel Bead kit v3.1 and were processed with the Chromium Controller (10X Genomics). Library for cDNA and cell surface markers were prepared. Sequencing was carried out using the Illumina NovaSeq technology (GenoBird, Nantes) on each 12 pools. Primary analysis was performed on each pool using the Cell ranger pipeline v5.0.0 (10X Genomics). Raw bcl files were demultiplexed with the FastQC v0.11.5. Count matrices were generated. Further analysis used bioinformatic tools Seurat v4.0 and R v4.1.
  • scRNAseq data processing
  • Quality control steps were applied to all 12 pools individually to assess for doublets and apoptosis. One pool of 3 samples was excluded from the experiment due to poor cell retention during the Chromium emulsion step. The remaining samples were loaded and integrated into one dataset. Data was normalized and scaled with the NormalizeData and ScaleData functions. Principal component analysis (PCA) was performed with RunPCA function. Dimensionality of this dataset was reduced using a Uniform Manifold Approximation and Projection (UMAP) algorithm to obtain a 2D representation of the cells according to the first 30 PCA components. The FindNeighbours and FindClusters functions were used with a resolution of 0.75 to cluster cells. The FindAllMarkers and Findmarkers functions were used to characterize and annotate these clusters. A subset of this PBMC dataset was made to study T cells more specifically. Differentially expressed genes were analyzed using Wilcoxon and DESeq2 statistical tests.
  • GSEA
  • GSEA (gene set enrichment analysis) was performed on lists of genes differentially expressed in clusters of interest, using the Seurat function EnrichR package on R, with references to the Kyoto encyclopaedia of genes and genomes (KEGG 2021), and gene ontology (GO 2021) databases.
  • Flow cytometry analysis
  • Frozen PBMC were thawed and stained with labelled antibodies against CD3 (BV711, clone : UCHT1), CD8 (BV605, clone : SK1), CD4 (V500, clone : RPA-T4), CD62L (BV650, clone : Dreg56), CCR7 (PE, clone : 3D12), CD45RA (FITC, clone : HI100), CD56 (BUV395, clone : B159), CD127 (BV373, clone : hIL-7R-M21), and CD94 (PerCP-Cy5.5, clone : HP-3D9) from BD Biosciences, as well as a labelled antibody against TCR Vα7.2 (PE-Cy7, clone : 3C10) from Biolegend. This staining was performed at 4°C in staining buffer (PBS, penicillin-streptomycin, 5% BSA, 2mM EDTA). Antibodies were added sequentially, starting with anti-CCR7 for 10 min. The other antibodies were then added for 20 min. Viability staining was finally performed with DAPI in phosphate saline buffer (PBS) at 4°C for 20 min. Fluorescence was analyzed on a BD FACSCelesta™ cell analyser with BD FACSDiva software (BD Biosciences).
  • sGFAP and sNfL measurements
  • MS patients from the OFSEP cohort were also tested for various parameters during diagnosis and follow-up visits, including serum glial acidic fibrillary protein (sGFAP), serum neurofilament light (sNfL) chain. Serum NfL concentrations were prospectively determined with the SimoaTM Human Neurology 4-Plex “A” kit (Quanterix Corp, Boston, MA), according to the manufacturers’ instructions. SimoaTM was calibrated using the provided standards. All samples were measured in simplicate, on the same day, after a single thaw, with a 1:4 dilution. The lower limit of detection was 0.104 pg/ml and 0.221 pg/ml for NFL and GFAP, respectively.
  • Cell Sorting for transcriptomics study:
  • Peripheral Blood Mononuclear cells (PBMC) were isolated using a standard Ficoll gradient according to manufacturer’s instructions (Eurobio, Les Ulis, France) and frozen until used. Cells were thawed and stained with labeled antibodies against CD3 (anti-CD3-PE, clone: UCHT1, BD biosciences), CD8 (anti-CD8-APC-H7, clone: SK1, BD Biosciences) , CD45RA (anti-CD45RA-APC-Vio770, clone: T6D11, Miltenyi biotec), Vα7.2 (anti-Vα7.2-FITC, clone: 3C10, Biolegend) and DAPI. Live non-MAIT memory CD8+ T cells (CD3+CD8+CD45RA-Vα7.2-DAPI-) were sorted using the BD ARIA FACS II cell sorter. Purity of sorted cells from PBMC was always greater than 96%.
  • Capture of single-cells and target genes pre-amplification
  • Each single-cell was isolated using the Fluidigm C1 system and C1 integrated fluidic circuits (IFC_5-10µm). The C1 experiments were performed according to the manufacturer’s instructions. Briefly, sorted live CD3+CD8+CD45RA-Vα7.2- cells were resuspended at a concentration of 330,000 PBMC/mL of culture medium (RPMI1640 10%FBS Penicilin-Streptomycin and glutamine). IFC was primed and then cells loaded into the chip. After the cells loading, the chip was imaged to confirmed the single-cells capture in each well (wells with 2 or more cells were not analyzed with the Biomark systems). Following the cell loading, the lysis final mix, the reverse transcription final mix and the preamp final mix were prepared using the Single Cell-to-Ct Kit (Ambion-Thermo Fisher Scientific). The preamp Final Mix contain a pool of 96 Taqman® probes (Thermo Fisher Scientific) at a concentration of 180nM (Supplemental Table 5 for Taqman® probes reference), 36 forward primers (Set 1)(180nM) and 1 reverse primer Cb1 (180nM) for the Vβ chain of the TCR (sequences are given in Supplemental Table 4). Lysis, reverse transcription and pre-amplification mix were loaded in the Fluidigm C1 device and amplified products were harvested the next morning.
  • Quantitative PCR
  • Following the C1 sample harvesting, we performed a first qPCR on a StepOnePlus instrument (Thermo Fisher Scientific) to confirmed presence of CD8+ T cells using the CD8 Taqman® probe (already used for the C1 preamplification). The 96 genes expression analysis of the preamplified amplicons was then performed with a 96.96 Dynamic Array® IFC using a Biomark HD (Fluidigm). This chip allows gene expression analysis of 96 genes in 96 single-cells simultaneously. The Biomark experiments were performed according to manufacturer’s recommendation. Briefly, the chip was first primed, then the 96 samples and the 96 Taqman probes were loaded in the chip using the fluidigm device. Finally, the qPCR amplification was performed in the Biomark apparatus. Data were then collected to be analyzed.
  • Statistical analyses
  • Statistical analyses were performed using GraphPad Prism v8.0.2 , excel and R v4.1. For the study cohort, proportions were analyzed using Fisher’s exact test for two groups or a chi-squared test followed by a residual analysis for more than two groups. For the validation cohort, Mann-Whitney and Welch’s t tests were used to compare study groups. ROC (receiver operating characteristic curve) analyses were used to test the ability of chosen parameters to discriminate between patient study groups. Linear regressions were used to assess correlation between chosen parameters and disease severity. Statistically significant results were marked according to standard notions with *p < 0.05, **p < 0.01 and ***p < 0.001.
  • Results
    1. scRNAseq analyses of T cells revealed differential gene expression between patients with multiple sclerosis and healthy controls.
  • Patients from groups 1 and 2 were recruited while treatment naïve during their first demyelinating event. A third group of healthy volunteers was added as controls. Blood samples were processed to isolate PBMCs, which were then sequenced together in pools of three samples ( ). scRNA sequencing with the 3’ CITE-seq technique yielded a total of 137741 blood single-cell transcriptomes. After filtering and normalization, 92396 cells were present in this dataset. Cells were clustered according to their similarities and annotated ( ). This dataset contained total PBMCs, however T cell clusters were isolated and re-clustered into a separate dataset, with 15 final clusters ( ). We used the ADT library for surface markers: CCR7, CD45RA, CD56, TCR Vα7.2. These antibodies help determine naïve and memory populations as well as determine MAIT cells that express the invariant TCR Vα7.2 and NK cells. The inventors identified 9 clusters of CD4+ T cells : two naïve CD4+ cell clusters (CD4n1 and CD4n2), 4 effector memory CD4+ cell clusters (CD4em1 to CD4em4), regulatory CD4+ T cells (Tregs), as well as two other small CD4+ clusters (CD4a and CD4b). Five CD8+ cell clusters were identified, composed of one naïve CD8+ cell cluster (CD8n), two effector memory CD8+ cell clusters (CD8em1 and CD8em2), MAIT cells and NKT cells. Additionally, one remaining cluster (Tgd) expressed neither CD4 nor CD8 genes but presented markers often found in gamma-delta T cells.
  • Distribution of clusters among MS patients and controls was determined and, in this study, we compared T cell cluster distribution between MS patients and HV ( ). Two CD4+ T cell clusters (CD4em1 and CD4em2) were increased in the blood of MS patients compared to HV. The cluster of CD8+ naïve T cells was also increased in MS patients compared to HV. NKT cells and CD8 effector memory cells from the CD8em1 cluster were increased in HV individuals compared to MS patients.
    1. scRNAseq analyses of T cells revealed differential gene expression between patients with aggressive and non-aggressive disease evolution at 2 years.
  • While comparing early MS patients to controls is of interest, inventor’s main goal remains to identify cells linked specifically to disease severity. Distribution of T cell clusters was also analyzed for the two MS study groups. In this case, there was no significant difference in distribution of CD4+ T cell clusters between the MS patient groups. However, two clusters of CD8+ T cells were found increased in the blood of patients with severe MS : cluster 6 (CD8em1 : NK-like CD8+ T cells) and cluster 11 (NKT cells) ( ). Both clusters contain memory cells. Cells from the CD8em1 cluster have an effector memory phenotype (CCR7- CD45RAlow), express migration genes (ITGA4), cytotoxicity genes (PRF1, GZMA, GZMB, GNLY) and NK genes (KLRD1, NKG7) as shown on and called NK-like CD8+ T cells thereafter. These cells -appear to be migratory cells capable of cytotoxicity. Further studies were performed to assess the differences in gene expression of the cells between the two patient groups. Apart from ribosomal gene RPS26, no genes had a significant difference in expression between CD8em1 cells of aggressive MS and non-aggressive MS patients ( ). The major pathways involved in cells from the CD8em1 cluster were linked to antigen processing and presentation, immune diseases, and pro-inflammation through GSEA ( ).
    1. Confirmation on a multicent er validation cohort by flow cytometry that NK-like CD8+ T cell frequency increases with aggressive MS
  • The inventors next aimed to confirm the increase in NK-like CD8+ T cells in the blood of patients who will develop aggressive MS at the protein level on a larger cohort. We used flow cytometry with antibodies targeting markers revealed to be expressed by NK-like CD8+ T cells during the transcriptomic analysis. PBMCs were collected from the blood of 60 patients with multiple sclerosis during their first demyelinating event (31 patients from Group 1 and 29 patients from Group 2) as well as 30 healthy controls. The following surface markers were targeted to identify NK-like CD8+ T cells : CD3, CD56, CD4, CD8, CD45RA, CCR7, TCR Vα7.2, CD62L, CD127, CD94 and a viability marker. NK-like CD8+ T cells are CD3+ CD56- CD4- CD8+ CD45RAlow CCR7- TCR Vα7.2- CD62L- CD127- CD94+ ( ). NK-like CD8+ T cells were found to be increased in the blood of patients who later develop aggressive MS compared to those who do not, thus confirming our findings from the scRNAseq dataset ( ). We also studied effector memory CD4+ T cells and naïve CD8+ T cells as they were increased in MS patients compared to controls in the transcriptomic study. This cytometry assay confirms increase of effector memory CD4+ T cells at the protein level in the blood of MS patients compared to controls ( ). NKT were not found to be increased in patients with severe MS.
    1. N K -like CD8+ T cell frequency discriminates patients with different evolutions at disease onset
  • As NK-like CD8+ T cells appear to be increased at onset in patients evolving 2 years later towards a severe MS, the inventors also looked at other known factors associated with severe MS to determine if combining them with NK-like frequency helped determine disease severity more accurately. Optic neuritis as a sole first demyelinating event is known to be a good prognosis factor for MS. However, there was no difference in the number of appearances of optic neuritis as a first demyelinating event between the two MS groups (data non shown). Lesion burden is also known to be associated with a poor prognosis. We indeed found that patients from Group 1 with aggressive MS were more likely to have a high lesion burden (> 9 lesions) at onset, compared to patients from Group 2 ( ). With an area under the curve (AUC) of 0.673 ( ), NK-like CD8+ T cell frequency seems to be a more accurate parameter to monitor disease severity than lesion burden where the AUC is 0.607.
  • Using the clinical information available for the patients in the cytometry validation cohort, the inventors also compared the frequency of NK-like CD8+ T cells among groups of patients divided by various parameters other than the choice of treatment administered at 2 years after onset. Patients were divided into group according to : 1) the detection of gadolinium enhancing lesions or lack thereof at onset, 2) the presence of spinal cord lesions or lack thereof at onset, 3) the combination of a high EDSS (≥3) and pyramidal signs at onset compared to others ( ) as these factors were reported as prognostic factor for a severe MS at disease onset. Frequency of NK-like CD8+ T cells was increased in patients with spinal cord lesions at onset compared to those without and in patients with a high EDSS (≥3) and pyramidal signs at onset compared to those without. NK-like CD8+ T cell frequency was not statistically different in the other groups of patients compared (). New lesion at 1 year and 2 years is more frequent in aggressive MS group compared to non-aggressive group and also EDA (Evidence of Disease Activity) is more frequent in the Aggressive MS group compared to non-aggressive group ( ). Sensitivity and sensibility analyses showed that a combination of NK-like CD8+ T cell frequency at onset, lesion burden at 1 year, and EDA at 1 year increased predictive value for aggressive MS up to an AUC of 0.822 ( ).
  • Serum GFAP and sNfL levels were also higher in patients who develop aggressive MS than patients who do not (Figures 17 and 18 respectively). Combining NK-like CD8+ T cell frequency, sGFAP and sNfL levels yielded a higher predictive value for aggressive MS than each factor separately (AUC = 0.843) ( ).
    1. N K -like CD8+ T cell s are associated with MS activity
  • The High-throughput qPCR data allow the inventor to identify the NK-like CD8+ T cell subset that is significantly enriched in cells coming from MS patients during a relapse episode (cluster E) ( ). In addition, flow cytometry analysis shows that there is a correlation between the frequency of NK-like CD8+ T cells and the time elapse since the relapse episode, more specifically, the closer the relapse, the higher the frequency of NK-like CD8+ T cells ( )
  • Discussion
  • In an autoimmune disease like multiple sclerosis, administering the appropriate treatment as early as possible is crucial for disease outcome. However, choosing the right treatment at onset remains a challenge. We aimed to reveal new biomarkers to help determine disease prognosis with the OutcoMS project. Focusing this study on CD8+ T cells as they are one of the main immune cell populations infiltrating the CNS and found in MS lesions, we identified a population of CD8+ T cells through single-cell transcriptomics that is significantly increased in the blood of CIS patients who later develop aggressive MS compared to those who later develop a non-aggressive form of the disease.
  • Sensitivity is low when at 80% specificity, meaning that this test could have false negatives and fail correctly prognose aggressive MS. However, a threshold at 80% sensitivity, while allowing for false positives, would make sure most patients with severe MS are correctly identified. We can argue that since they already have MS, it is better to correctly treat the most people than to minimize over-treatment. Furthermore, when combined with serum Nfl and GFAP, the prognostic value of NK-like CD8+ T cell frequency in regard to aggressive MS significantly increases.
  • Interestingly, NK-like CD8+ T cells were shown to exert suppressive activity with a similar molecular signature to the NK-like CD8+ T cells identified in this study. While our team demonstrates the cells have notable cytotoxic capacities, further studies are needed to elucidate their exact function and role in MS.
    •

Claims (13)

  1. An in vitro prognosis method of an aggressive multiple sclerosis in a patient afflicted by a multiple sclerosis, the method comprising:
    - quantifying in a biological sample of the patient the amount of cells called NK-like cells having a phenotype CD3+, CD8+, CD45RA-, Vα7.2-, CCR7-, and CD94+, preferably cells having a phenotype CD3+, CD8+, CD4-, CD45RA-, CCR7-, CD94+, CD27-, CD62L-, CD127- and CD56-; in order to obtain quantified amount of NK-like cells,
    - comparing the quantified amount of NK-like cells with the control amount of a NK-like cell in a control biological sample, and
    - concluding that
    - if the quantified amount of NK-like cells is lower than or equal to the control amount, then the patient is not liable to develop an aggressive multiple sclerosis, and
    - if the quantified amount of NK-like cells is higher than 0.21% of total CD8 T cells, the method gives a sensitivity of 80% and a specificity of 38% to detect an aggressive multiple sclerosis within 24 months or more.
  2. The method according to claim 1, the method comprising:
    - quantifying in a biological sample of the patient the amount of cells called NK-like cells having a phenotype CD3+, CD8+, CD45RA-, Vα7.2-, CCR7-, and CD94+, preferably cells having a phenotype CD3+, CD8+, CD4-, CD45RA-, CCR7-, CD94+, CD27-, CD62L-, CD127- and CD56-; in order to obtain quantified amount of NK-like cells,
    - comparing the quantified amount of NK-like cells with the control amount of a NK-like cell in a control biological sample, and
    - concluding that
    - if the quantified amount of NK-like cells is lower than or equal to the control amount, then the patient is not liable to develop an aggressive multiple sclerosis, and
    - if the quantified amount of NK-like cells is higher than 0.51% of total CD8 T cells, the method gives a sensitivity of 34% and a specificity of 80% to detect an aggressive multiple sclerosis within 24 months or more.
  3. The method according to claim 1 or 2, wherein said NK-like cell, upon IL-15 stimulation expresses KIR and CD56 markers.
  4. The method according to anyone of claims 1 to 3, wherein aggressive multiple sclerosis is multiple sclerosis originating from multiple sclerosis having spinal cord lesions at onset and multiple sclerosis with a high Expanded Disability Status Scale equal to or higher than 3 and pyramidal signs at onset.
  5. An isolated and purified NK-like cell having a phenotype CD3+, CD8+, CD4-, CD45RA-, CCR7-, CD94+, CD27-, CD62L-, CD127- and CD56-.
  6. The isolated and purified NK-like cell as defined in claim 5, for its use as medicament.
  7. Use of a NK-like cell having a phenotype CD3+, CD8+, CD45RA-, Vα7.2-, CCR7-, and CD94+ v, for carrying out a method for prognosing the likelihood to develop a relapse of multiple sclerosis, or to develop an aggressive multiple sclerosis.
  8. The use according to claim 7, wherein the NK-like cell having a phenotype CD3+, CD8+, CD4-, CD45RA-, CCR7-, CD94+, CD27-, CD62L-, CD127- and CD56-.
  9. The use according to claim 7 or 8, wherein said NK-like cell, upon IL-15 stimulation expresses KIR and CD56 markers.
  10. A kit consisting essentially of means for detecting, at the membrane of a cell, the following markers: CD3, CD8, CD45RA, Vα7.2, CCR7, and CD94, preferably the markers CD3, CD8, CD4, CD45RA, CCR7, CD94, CD27, CD62L, CD127 and CD56.
  11. The kit according to claim 10, consisting of:
    - means for detecting, at the membrane of a cell, the following markers: CD3, CD8, CD45RA, Vα7.2, CCR7, and CD94, possibly CD3, CD8, CD45RA, Vα7.2, CCR7, CD94, KIR and CD56 and,
    - a biological sample comprising cells of an individual known to not having developed a relapse of multiple sclerosis or an aggressive multiple sclerosis.
  12. A compound inhibiting the cytotoxic activity of NK-like cell having a phenotype CD3+, CD8+, CD4-, CD45RA-, Vα7.2-, CCR7-, CD94+, CD27-, CD62L-, CD127- and CD56-, as a drug.
  13. The compound according to claim 12, for its us for treating relapse of multiple sclerosis or a severe multiple sclerosis.
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