WO2018220231A1

WO2018220231A1 - Method for monitoring and/or predicting the efficacy of treatment in a multiple sclerosis patient

Info

Publication number: WO2018220231A1
Application number: PCT/EP2018/064645
Authority: WO
Inventors: Luisa María VILLAR GUIMERANS; José Carlos ÁLVAREZ CERMEÑO; Lucienne COSTA-FROSSARD FRANCA; Susana SAINZ DE LA MAZA CANTERO; Ernesto ROLDÁN SANTIAGO; Noelia VILLARRUBIA MIGALLÓN; Silvia MEDINA HERAS
Original assignee: Fundación Para La Investigación Biomédica Del Hospital Universitario Ramón Y Cajal
Priority date: 2017-06-02
Filing date: 2018-06-04
Publication date: 2018-12-06
Also published as: EP3631463A1

Abstract

The present invention provides a new method for determining whether a dimethyl fumarate 10 treatment is effective for a particular Multiple Sclerosis (MS) patient.A pro-tolerogenic shift in the blood leukocyte profile associates with an optimal response to DMF in(relapse remitting MS)RRMS patients. This method and the kit to perform it, allowsfor the identification ofgood responders to DMF treatment once drug administration has been initiated.

Description

METHOD FOR MONITORING AND/OR PREDICTING THE EFFICACY OF TREATMENT IN A

MULTIPLE SCLEROSIS PATIENT

Technical field

The present invention can be included in the field of medicine. Specifically, the present application relates to a new method for determining whether a dimethyl fumarate treatment is effective for a particular Multiple Sclerosis (MS) patient.

Background art

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) of unknown etiology, in which immune cells migrate to the central nervous system inducing demyelination and axonal damage, the main cause of disability in the disease¹. MS affects approximately 2.5 million individuals worldwide and is the most common demyelinating disease affecting young adults. Different therapeutic options are available to ameliorate disease activity in MS patients^2,3. Unfortunately, the response to treatment is not always satisfactory and a number of suboptimal responders are found for every disease modifying treatment used in MS.

Dimethyl fumarate (DMF; Tecfidera®, Biogen, Cambridge, MA), is an approved treatment for relapsing remitting MS (RRMS). The efficacy of oral DMF in treating RRMS in adults was evaluated in two large 2-year double-blind, multinational, phase III trials, DEFINE⁴ and CONFIRM⁵ and their dose-blind extension, ENDORSE.^6,7 DMF reduces relapse rate by about 50% and both the number of new or enlarging hyper intense lesions on T2-weighted images and the number of gadolinium-enhancing lesions. DMF mechanism of action has not been fully elucidated, although it was postulated that it plays both antioxidant and immunomodulatory effects, including a reduction in cytokine production⁸, in the migratory capacity of the immune cells by inhibition of nuclear factor kappa B (NF-κΒ)⁹ and the activation of the nuclear factor erytroid 2-related factor 2(Nrf2) transcriptional pathway.¹⁰

It was also suggested that it affects predominantly memory T and B cells, and induces a shift towards anti-inflammatory response.^11"14 However, data from immunological changes in RRMS patients treated with DMF are still sparse and more comprehensive insights on its mechanism of action are needed.

No evidence of disease activity (NEDA) is defined as the absence of new relapses, disability progression and magnetic resonance imaging (MRI) activity on a disease modifying treatment.¹⁵Patients achieving NEDA in the first years of treatment have a high probability of remaining free of progression for long¹⁶. Thus, the finding of biomarkers predicting this status at the beginning of a treatment would be very useful for therapeutic decisions. Thus, it is an objective of the present invention to provide a method for monitoring and/or predicting the efficacy of a DMF treatment in patients who suffer from MS.

Figures Figure 1 : CD4 (A and B) and CD8 (C and D) T cells from a patient with optimal response to dimethyl fumarate at basal state (A and C) and after six months of treatment with DMF (B and D). Naive (N), central memory (CM), effector memory (EM) and terminally differentiated cells (TD) were studied according to their differential membrane expression of CD197 (CCR7) and CD45RO. Plots A and B are gated on total CD4+ T cells. Plots C and D are gated on total CD8+ T cells. Percentages showed in plots are referred to total peripheral blood mononuclear cells.

Figure 2: Dot plots showing T cells producing interferon gamma (IFN-γ, A and B) and B cells producing tumor necrosis factor-alpha (TNF-a, C and D) from a patient showing optimal response to dimethyl fumarate at basal state (A and C) and after six months of treatment with DMF (B and D). Plots A and B are gated on total T cells. Plots C and Dare gated on total B cells. Percentages showed in the plots are referred to total peripheral blood mononuclear cells.

Summary of the invention

The present invention provides a method for monitoring and/or predicting the efficacy of a dimethyl fumarate treatment in a patient who suffers from Multiple Sclerosis, comprising the steps of:(a) isolating peripheral blood mononuclear cells from isolated blood samples obtained before and after dimethyl fumarate treatment; and (b) quantifying the amount of cells of one or more peripheral blood mononuclear cell subset in the before and after sample. Further, the present invention provides a kit comprising antibodies for identifying CD4+ central memory cells, CD8+ T cells, CD8+ central memory cells, natural killer T cells, memory B cells, CD56bright natural killer cells, CD4+IFN-V+ T cells, CD8+IFN-V+ T cells, CD8+TNF-a+ T cells, CD8+GM-CSF+ T cells and/or CD19+TNF-a+ B cells, and its use with the method of the present invention. Detailed description of the invention

Definitions

The terms "treatment and "therapy", as used in the present application, refer to a set of hygienic, pharmacological, surgical and/or physical means used with the intent to cure and/or alleviate a disease and/or symptoms with the goal of remediating the health problem. The terms "treatment' and "therapy" include preventive and curative methods, since both are directed to the maintenance and/or reestablishment of the health of an individual or animal. Regardless of the origin of the symptoms, disease and disability, the administration of a suitable medicament to alleviate and/or cure a health problem should be interpreted as a form of treatment or therapy within the context of this application.

The terms "individuaf, "patient or "subject are used interchangeably in the present application and are not meant to be limiting in any way. The "individuaf, "patient or "subject can be of any age, sex and physical condition.

The terms "Multiple Sclerosis" or "MS" refer to a chronic inflammatory disease of the central nervous system (CNS) of unknown etiology, in which immune cells migrate to the central nervous system inducing demyelination and axonal damage.

The terms "relapse remitting Multiple Sclerosis" or "RRMS" refer to a type of MS characterized by flare-ups or relapses. The flare-ups or relapses may occur after a period of inactivity of the disease. The terms "Peripheral blood mononuclear cell' or "PBMC" refer to any peripheral blood cell having a round nucleus.

The terms "Peripheral blood mononuclear cell subset or "PBMC subset refer to a specific type of PBMC characterized by expressing a particular surface antigen and/or cytokine.

The term "surface antigen" refers to any membrane-bound target, usually a transmembrane protein, which can be used to characterize a PBMC and categorize the PBMC into a PBMC subset. Examples of surface antigens include: CD4, CD8, CCR7, CD45RO, CD3, CD25, CD127, CD19, CD38, CD24 and CD56.

The term "cytokine" refers to a broad class of small proteins that are important in cell signaling. Examples of cytokines include interleukin-10 (IL-10), interferon-γ (IFN-γ), granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-a) and interleukin-17 (IL-17).

The term "isolated blood sample" refers to any blood sample which has been previously obtained, i.e. isolated, from a patient. It is to be understood that the act of obtaining the blood samples from a patient is not an aspect of the present invention. Method

The present invention provides a method for monitoring and/or predicting the efficacy of a dimethyl fumarate treatment in a patient who suffers from Multiple Sclerosis, comprising the steps of:(a) isolating peripheral blood mononuclear cells from isolated blood samples obtained before and after dimethyl fumarate treatment; and (b) quantifying the amount of cells of one or more peripheral blood mononuclear cell subset in the before and after sample.

According to their differential antigen expression, CD4+ and CD8+ T cells were classified as: naive (CCR7+ CD45RO-); central memory (CM) (CCR7+ CD45RO+); effector memory (EM) (CCR7- CD45RO+); terminally differentiated (TD) (CCR7- CD45RO-). Regulatory CD4 T cells (Treg) were defined as CD3+ CD4+ CD25hi CD127low. B cells were classified as: memory (CD19+ CD27dim CD38dim), plasmablasts (CD19+ CD27hi CD38hi); and regulatory (Breg) (CD19+ CD27- CD24hi CD38hi or CD19+ IL-10+). Further, natural killers cells were classified as natural killer (NK) cells (CD56dim CD3-), natural killer T (NKT) cells (CD56dim CD3+), and CD56bright NK cells (CD3- CD56bright). CD4+ and CD8+ T cells were also classified by their intracellular cytokine production. Cells could be CD4+ or CD8+ as well as positive or negative for IL-10, IFN-Y, GM-CSF, TNF-a and/or IL-17. The PBMC subsets which were up- or down-regulated when comparing patients where the DMF treatment was effective versus those patients where the DMF treatment was ineffective were CD4+ central memory cells, CD8+ T cells, CD8+ central memory cells, natural killer T cells, memory B cells, CD56bright natural killer cells, CD4+IFN-V+ T cells, CD8+IFN-V+ T cells, CD8+TNF-Q+ T cells, CD8+GM-CSF+ T cells and/or CD19+TNF-Q+ B cells.

First results showed significant changes in immune cell subset expression profile (effector, regulatory and memory cells) after 6 months of treatment with DMF. The expression of these different subsets was compared over time between NEDA and ODA (ongoing disease activity) subgroup of patients.

When changes in PBMC subsets between samples collected at time 0 and after 6 months of treatment were studied, both groups showed similar results in the blood lymphocyte profile. But, the blood lymphocyte profile changed after administration with DMF. In this case, both groups (NEDA and ODA) showed similar increases in naive CD4+ and CD8+T cells, and similar decreases in EM CD4+ T cells, total CD8+ T cells and in EM and TD CD8+ cells during treatment. The most important result is that NEDA patients showed a different profile for other lymphoid subset: decrement of CM CD4+ and CD8+ T cells, high decrease in NKT cells and memory B cells, and higher increment in CD56bright cells. In order to complete our understanding of immune profile changes, we studied the intracellular cytokine production in the same samples. There was a decrement in levels of CD4+ and CD8+ IFN-y+ T cells, CD4+ and CD8+ TNF-a T cells, CD8+ GM-CSF+ T cells and CD19+ TNF-a + B cells restricted in most cases only to NEDA group. These results strongly suggest that changes in immune profile in both cytokine production and percentage of cell subsets are associated with optimal responses to DMF.

Therefore, in a preferred embodiment, the cells quantified in step (b) are CD4+ central memory cells, CD8+ T cells, CD8+ central memory cells, natural killer T cells, memory B cells, CD56bright natural killer cells, CD4+IFN-V+ T cells, CD8+IFN-V+ T cells, CD8+TNF-a+ T cells, CD8+GM-CSF+ T cells and/or CD19+TNF-a+ B cells. In other words, the PBMC subset can be CD4+ central memory cells, CD8+ T cells, CD8+ central memory cells, natural killer T cells, memory B cells, CD56bright natural killer cells, CD4+IFN-V+ T cells, CD8+IFN-V+ T cells, CD8+TNF-Q+ T cells, CD8+GM-CSF+ T cells and/or CD19+TNF-Q+ B cells.

In a preferred embodiment, the method further comprises the step of: (c) assigning those patients who exhibit a: (i) decrease in the amount of CD4+ central memory cells, CD8+ T cells, CD8+ central memory cells, natural killer T cells and/or memory B cells; (ii) an increase in the amount of CD56bright natural killer cells; and/or (iii) a decrease in CD4+IFN-y+ T cells, CD8+IFN-Y+ T cells, CD8+TNF-Q+ T cells, CD8+GM-CSF+ T cells, and/or CD19+TNF-Q+ B cells after dimethyl fumarate treatment to the group of patients who have a higher probability of responding positively and/or optimally to a dimethyl fumarate treatment.

In a more preferred embodiment, the method further comprises the step of: (c) assigning those patients who exhibit a: (i) decrease in the amount of CD4+ central memory cells and/or CD8+ central memory cells; and/or (ii) a decrease in CD4+IFN-y+ T cells, CD8+IFN-y+ T cells, CD8+GM-CSF+ T cells, and/or CD19+TNF-a+ B cells after dimethyl fumarate treatment to the group of patients who have a higher probability of responding positively and/or optimally to a dimethyl fumarate treatment.

As discussed in the Examples, a decrease in the amount of CD4+ central memory cells, CD8+ T cells, CD8+ central memory cells, natural killer T cells and/or memory B cells or an increase in the amount of CD56bright natural killer cells was correlated with effective treatment. A decrease in CD4+IFN-Y+ T cells, CD8+IFN-V+ T cells, CD8+TNF-Q+ T cells, CD8+GM-CSF+ T cells, or CD19+TNF-Q+ B cells was also correlated with effective treatment.

In a preferred embodiment, the method for monitoring and/or predicting the efficacy of a DMF treatment in a patient who suffers from MS is a method for monitoring and/or predicting the efficacy of a DMF treatment in a patient who suffers from RRMS. In a preferred embodiment, MS is RRMS. It is a goal to be able to predict whether DMF therapy will be effective for a patient. To achieve this, a sample of blood is taken from the patient before the DMF treatment begins and stored as a reference sample. Then, the patient is treated with DMF for a certain amount of time before another sample is taken. The PBMCs are then isolated from the sample and reference sample. And, the amount of cells in certain PBMC subsets are quantified and compared between samples. The present invention shows that certain subsets are up- or down-regulated if the treatment was effective. Therefore, one could use the present invention to predict whether the treatment will be effective and/or the present invention could be used to monitor whether the treatment is effective. Previous approaches relied on clinical and MRI activity after one year of follow-up to determine whether a treatment is effective or is going to be effective. The present invention expedites that process by increasing the predictive power by at least 6 months. Further, the present invention provides a more accurate determination on whether the DMF treatment is or will be effective. In a preferred embodiment, the isolated blood samples were obtained before and after a dimethyl fumarate treatment which lasted for at least 0, 1 , 2, 3, 4 or 5 months, preferably at least 6 months. In other words, a blood sample is obtained before DMF treatment. Then, the patient undergoes a treatment regime for at least 0, 1 , 2, 3, 4, or 5, preferably 6 months. Then, a blood sample is taken again. Both samples are then compared for up- or down-regulation of one or more PBMC subsets. In the case that the DMF treatment lasted 0 months, a sample is taken immediately after the first treatment with DMF or any time between the first treatment and a time period of less than 1 month.

It is also envisioned that the method of the present invention could be used to continuously monitor the efficacy of a therapy. Thus, multiple blood samples could be analyzed to obtain a time-dependent graph of the amount of one or more PBMC subsets.

As shown in the Examples, when the therapy was effective, the amount of CD56bright natural killer cells was negatively correlated with CD4+IFN-y+ T cells, CD8+IFN-y+ T cells and CD8+TNF-Q+.

Thus, in a preferred embodiment, the amount of CD56bright natural killer cells is correlated with the amount of CD4+IFN-V+ T cells, CD8+IFN-V+ T cells and/or CD8+TNF-a+ T cells and patients who exhibit a negative correlation between CD56bright natural killer cells and CD4+IFN-Y+ T cells, CD8+IFN-V+ T cells and/or CD8+TNF-a+ T cells after dimethyl fumarate treatment are assigned to the group of patients who have a higher probability of responding positively and/or optimally to a dimethyl fumarate treatment. In a preferred embodiment, the PBMCs are isolated from the isolated blood samples by using centrifugation. Preferably, the centrifugation is Ficoll density gradient centrifugation.

In the present Examples, cells were stained using anti-CD8-FITC, anti-CD27-FITC, anti-CD24- PE, anti-CD197-PE (CCR7-PE), anti-CD3-PerCP, anti-CD38-PE-Cy5.5, anti-CD19-PE-Cy7, anti-CD25-PE-Cy7, anti-CD45RO-APC, anti-CD56-APC, anti-CD4-APC-H7, anti-CD8-APC-H7, anti-CD3-BV421 , anti-CD127-BV421 and/or anti-CD45-V500 antibodies. Optionally, the cells were also permeabilized and stained with anti-IFNv-FITC, anti-TNF-a-PerCP-Cy5.5, anti-GM- CSF-PE, anti-IL-10-PE and/or anti-IL-17-APC antibodies. After the cells were stained, they were quantified using flow cytometry.

In a preferred embodiment, the amount of cells is quantified using flow cytometry. Preferably, the isolated peripheral blood mononuclear cells are stained using antibodies recognizing surface antigens and then quantified using flow cytometry. More preferably, the isolated peripheral blood mononuclear cells are stained using antibodies recognizing surface antigens, permeabilized, then stained intracellular^ with antibodies recognizing cytokines and then quantified using flow cytometry.

In a preferred embodiment, the antibodies recognizing surface antigens bind to one or more targets selected from the group consisting of CD8, CD27, CD24, CCR7, CD3, CD38, CD19, CD25, CD45RO, CD56, CD4, CD8, CD3, CD127 and CD45.

In a preferred embodiment, the antibodies recognizing cytokines bind to one or more targets selected from the group consisting of IFNy, TNF-a, GM-CSF, IL-10 and IL-17. Kit

The present invention provides a kit comprising antibodies for identifying CD4+ central memory cells, CD8+ T cells, CD8+ central memory cells, natural killer T cells, memory B cells, CD56bright natural killer cells, CD4+IFN-V+ T cells, CD8+IFN-V+ T cells, CD8+TNF-a+ T cells, CD8+GM-CSF+ T cells and/or CD19+TNF-a+ B cells.

In a preferred embodiment, the kit comprises: antibodies for identifying CD56bright natural killer cells and CD4+IFN-y+ T cells, antibodies for identifying CD56bright natural killer cells and CD8+IFN-Y+ T cells, and/or antibodies for identifying CD56bright natural killer cells and CD8+TNF-a+ T cells. In this kit, the quantity of the population of CD56bright natural killer cells can be negatively correlated with the amount of CD4+IFN-y+ T cells, CD8+IFN-y+ T cells and/or CD8+TNF-Q+ T cells in patients where the treatment is working or may work in the future. In a preferred embodiment, the antibodies used to identify CD4+ central memory cells bind to CD4, CCR7 and CD45RO, the antibodies used to identify CD8+ T cells bind to CD8, the antibodies used to identify CD8+ central memory cells bind to CD8, CCR7 and CD45RO, the antibodies used to identify natural killer T cells bind to CD56 and CD3, the antibodies used to identify memory B cells bind to CD19, CD27 and CD38, the antibodies used to identify CD56bright natural killer cells bind to CD56 and CD3, the antibodies used to identify CD4+IFN- Y+ T cells bind to CD4 and IFN-γ, the antibodies used to identify CD8+IFN-Y+ T cells bind to CD8 and IFN-γ, the antibodies used to identify CD8+TNF-a+ T cells bind to CD8 and TNF-a, the antibodies used to identify CD8+GM-CSF+ T cells bind to CD8 and GM-CSF, and/or the antibodies used to identify CD19+TNF-a+ B cells bind to CD19 and TNF-a.

Preferably, the antibodies are labeled. More preferably, the labeled antibodies used to identify CD4+ central memory cells are anti-CD4-APC-H7, anti-CCR7-PE and anti-CD45RO-APC, the labeled antibody used to identify CD8+ T cells is anti-CD8-APC-H7 and/or anti-CD8-FITC, the labeled antibodies used to identify CD8+ central memory cells are anti-CD8-APC-H7 or anti- CD8-FITC, anti-CCR7-PE and anti-CD45RO-APC, the labeled antibodies used to identify natural killer T cells are anti-CD56-APC, and anti-CD3-BV421 and/or anti-CD3-PerCP, the labeled antibodies used to identify memory B cells are anti-CD19-PE-Cy7, anti-CD27-FITC and anti-CD38-PE-Cy5.5, the labeled antibodies used to identify CD56bright natural killer cells are anti-CD56-APC, and anti-CD3-BV421 and/or anti-CD3-PerCP, the labeled antibodies used to identify CD4+IFN-Y+ T cells are anti-CD4-APC-H7 and anti-IFN-Y-FITC, the labeled antibodies used to identify CD8+IFN-Y+ T cells are anti-CD8-APC-H7 or anti-CD8-FITC, and anti-IFN-Y- FITC, the labeled antibodies used to identify CD8+TNF-a+ T cells are anti-CD8-APC-H7 or anti- CD8-FITC, and anti-TNF-a-PerCP-Cy5.5, the labeled antibodies used to identify CD8+GM- CSF+ T cells are anti-CD8-APC-H7 or anti-CD8-FITC, and anti-GM-CSF-PE, and/or the labeled antibodies used to identify CD19+TNF-a+ B cells are anti-CD19-PE-Cy7 and anti-TNF-a- PerCP-Cy5.5.

In an alternative embodiment, the kit which comprises antibodies used to identify CD4+ central memory cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD4, anti- CCR7 and anti-CD45RO, or several tubes or recipients comprising separately or in combination each of these antibodies, and (ii) optionally a washing or buffer solution. Preferably, the antibodies are labeled. More preferably, the anti-CD4 antibody is anti-CD4-APC-H7, anti-CCR7 is anti-CCR7-PE, and/or anti-CD45RO is anti-CD45RO-APC. In an alternative embodiment, the kit which comprises antibodies used to identify CD8+ T cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD8, and (ii) optionally a washing or buffer solution. Preferably, the antibodies are labeled. More preferably, the anti- CD8 antibody is anti-CD8-APC-H7 and/or anti-CD8-FITC.

In an alternative embodiment, the kit which comprises antibodies used to identify CD8+ central memory cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD8, anti- CCR7 and anti-CD45RO, or several tubes or recipients comprising separately or in combination each of these antibodies, and (ii) optionally a washing or buffer solution. Preferably, the antibodies are labeled. More preferably, the anti-CD8 antibody is anti-CD8-APC-H7 or anti- CD8-FITC, anti-CCR7 is anti-CCR7-PE, and/or anti-CD45RO is anti-CD45RO-APC. In an alternative embodiment, the kit which comprises antibodies used to identify natural killer T cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD56 and anti- CD3, or several tubes or recipients comprising separately or in combination each of these antibodies, and (ii) optionally a washing or buffer solution. Preferably, the antibodies are labeled. More preferably, the anti-CD56 antibody is anti-CD56-APC, and anti-CD3 is anti-CD3- BV421 and/or anti-CD3-PerCP.

In an alternative embodiment, the kit which comprises antibodies used to identify memory B cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD19, anti-CD27 and anti-CD38, or several tubes or recipients comprising separately or in combination each of these antibodies, and (ii) optionally a washing or buffer solution. Preferably, the antibodies are labeled. More preferably, the anti-CD19 antibody is anti-CD19-PE-Cy7, anti-CD27 is anti-CD27- FITC, and anti-CD38 is anti-CD38-PE-Cy5.5.

In an alternative embodiment, the kit which comprises antibodies used to identify CD56bright natural killer cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD56 and anti-CD3, or several tubes or recipients comprising separately or in combination each of these antibodies, and (ii) optionally a washing or buffer solution. Preferably, the antibodies are labeled. More preferably, the anti-CD56 antibody is anti-CD56-APC, and anti-CD3 is anti-CD3- BV421 and/or anti-CD3-PerCP.

In an alternative embodiment, the kit which comprises antibodies used to identify CD4+IFN-y+ T cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD4 and anti-IFN- Y, or several tubes or recipients comprising separately or in combination each of these antibodies, (ii) optionally a washing or buffer solution, and (iii) optionally means for the fixation and permeabilization of cells and/or a buffer to wash the cells and to dilute the anti-cytokine antibodies for staining. Preferably, the antibodies are labeled. More preferably, the anti-CD4 antibody is anti-CD4-APC-H7, and anti-IFN-γ is anti-IFN-v-FITC. In an alternative embodiment, the kit which comprises antibodies used to identify CD8+IFN-y+ T cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD8 and anti-IFN- Y, or several tubes or recipients comprising separately or in combination each of these antibodies, (ii) optionally a washing or buffer solution, and (iii) optionally means for the fixation and permeabilization of cells and/or a buffer to wash the cells and to dilute the anti-cytokine antibodies for staining. Preferably, the antibodies are labeled. More preferably, the anti-CD8 antibody is anti-CD8-APC-H7 and/or anti-CD8-FITC, and anti-IFN-γ is anti-IFN-v-FITC.

In an alternative embodiment, the kit which comprises antibodies used to identify CD8+TNF-a+ T cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD8 and anti- TNF-a, or several tubes or recipients comprising separately or in combination each of these antibodies, (ii) optionally a washing or buffer solution, and (iii) optionally means for the fixation and permeabilization of cells and/or a buffer to wash the cells and to dilute the anti-cytokine antibodies for staining. Preferably, the antibodies are labeled. More preferably, the anti-CD8 antibody is anti-CD8-APC-H7 and/or anti-CD8-FITC, and anti-TNF-a is anti-TNF-a-PerCP- Cy5.5.

In an alternative embodiment, the kit which comprises antibodies used to identify CD8+GM- CSF+ T cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD8 and anti-GM-CSF, or several tubes or recipients comprising separately or in combination each of these antibodies, (ii) optionally a washing or buffer solution, and (iii) optionally means for the fixation and permeabilization of cells and/or a buffer to wash the cells and to dilute the anti- cytokine antibodies for staining. Preferably, the antibodies are labeled. More preferably, the anti-CD8 antibody is anti-CD8-APC-H7 and/or anti-CD8-FITC, and anti-GM-CSF is anti-GM- CSF-PE.

In an alternative embodiment, the kit which comprises antibodies used to identify CD19+TNF-a+ B cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD19 and anti- TNF-a, or several tubes or recipients comprising separately or in combination each of these antibodies, (ii) optionally a washing or buffer solution, and (iii) optionally means for the fixation and permeabilization of cells and/or a buffer to wash the cells and to dilute the anti-cytokine antibodies for staining. Preferably, the antibodies are labeled. More preferably, the anti-CD19 antibody is anti-CD19-PE-Cy7, and anti-TNF-α is anti-TNF-a-PerCP-Cy5.5.

In a preferred embodiment, the kit which comprises antibodies for identifying CD56bright natural killer cells and CD4+IFN-y+ T cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD56, anti-CD3, anti-CD4 and anti-IFN-γ, or several tubes or recipients comprising separately or in combination each of these antibodies, (ii) optionally a washing or buffer solution, and (iii) optionally means for the fixation and permeabilization of cells and/or a buffer to wash the cells and to dilute the anti-cytokine antibodies for staining. Preferably, the antibodies are labeled. More preferably, the anti-CD4 antibody is anti-CD4-APC-H7, anti-IFN-γ is anti-IFN-Y-FITC, the anti-CD56 antibody is anti-CD56-APC, and anti-CD3 is anti-CD3-BV421 and/or anti-CD3-PerCP.

In a preferred embodiment, the kit which comprises antibodies for identifying CD56bright natural killer cells and CD8+IFN-y+ T cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD56, anti-CD3, anti-CD8 and anti-IFN-γ, or several tubes or recipients comprising separately or in combination each of these antibodies, (ii) optionally a washing or buffer solution, and (iii) optionally means for the fixation and permeabilization of cells and/or a buffer to wash the cells and to dilute the anti-cytokine antibodies for staining. Preferably, the antibodies are labeled. More preferably, the anti-CD8 antibody is anti-CD8-APC-H7 and/or anti- CD8-FITC, anti-IFN-Y is anti-IFN-Y-FITC, the anti-CD56 antibody is anti-CD56-APC, and anti- CD3 is anti-CD3-BV421 and/or anti-CD3-PerCP.

In a preferred embodiment, the kit which comprises antibodies for identifying CD56bright natural killer cells and CD8+TNF-a+ T cells comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD56, anti-CD3, anti-CD8 and anti-TNF-a, or several tubes or recipients comprising separately or in combination each of these antibodies, (ii) optionally a washing or buffer solution, and (iii) optionally means for the fixation and permeabilization of cells and/or a buffer to wash the cells and to dilute the anti-cytokine antibodies for staining. Preferably, the antibodies are labeled. More preferably, the anti-CD8 antibody is anti-CD8-APC-H7 and/or anti- CD8-FITC, anti-TNF-a is anti-TNF-a-PerCP-Cy5.5, the anti-CD56 antibody is anti-CD56-APC, and anti-CD3 is anti-CD3-BV421 and/or anti-CD3-PerCP.

In a preferred embodiment, the kit comprises antibodies for identifying CD4+ central memory cells, CD8+ T cells, CD8+ central memory cells, natural killer T cells, memory B cells and CD56bright natural killer cells. Preferably, the kit comprises (i) a tube or recipient comprising monoclonal antibodies anti-CD4, anti-CCR7, anti-CD45RO anti-CD8, anti-CD56, anti-CD3, anti- CD19, anti-CD27, and anti-CD38, or several tubes or recipients comprising separately or in combination each of these antibodies, and (ii) optionally a washing or buffer solution. More preferably, the antibodies are labeled. Most preferably, the anti-CD4 antibody is anti-CD4-APC- H7, anti-CCR7 is anti-CCR7-PE, anti-CD45RO is anti-CD45RO-APC, anti-CD8 is anti-CD8- APC-H7 and/or anti-CD8-FITC, anti-CD56 antibody is anti-CD56-APC, anti-CD3 is anti-CD3- BV421 or anti-CD3-PerCP, anti-CD19 antibody is anti-CD19-PE-Cy7, anti-CD27 is anti-CD27- FITC, and/or anti-CD38 is anti-CD38-PE-Cy5.5. In a preferred embodiment, the kit comprises antibodies for identifying CD4+IFN-y+ T cells, CD8+IFN-Y+ T cells, CD8+TNF-Q+ T cells, CD8+GM-CSF+ T cells and/or CD19+TNF-Q+ B cells. Preferably, the kit comprises: (i) a tube or recipient comprising monoclonal antibodies anti- CD4, anti-IFN-Y, anti-CD8, anti-TNF-a, anti-GM-CSF, and anti-CD19, or several tubes or recipients comprising separately or in combination each of these antibodies, (ii) optionally a washing or buffer solution, and (iii) optionally means for the fixation and permeabilization of cells and/or a buffer to wash the cells and to dilute the anti-cytokine antibodies for staining. More preferably, the antibodies are labeled. Most preferably, the anti-CD4 antibody is anti-CD4-APC- H7, anti-IFN-Y is anti-IFN-Y-FITC, anti-CD8 antibody is anti-CD8-APC-H7 and/or anti-CD8-FITC, anti-TNF-a is anti-TNF-a-PerCP-Cy5.5, anti-GM-CSF is anti-GM-CSF-PE, and/or anti-CD19 antibody is anti-CD19-PE-Cy7.

The kit may further comprise any reagents (buffers, solutions, dyes...) needed to implement the method of the invention such as for example, reagents for sample washing, lysing and staining. Any one of the embodiments of the kit may further comprise dimethyl fumarate. Preferably, a separate tube or recipient comprising dimethyl fumarate.

It is particularly noted that different markers can be used to label the antibodies. However, preferred markers are PE (phycoerythrin), Cy7 (Cyanine 7), Cy5.5 (Cyanine 5.5), APC (allophycocyanin), H7, FITC (fluorescein isothiocyanate), BV421 (Brilliant Violet 421 ), V500 (BD Horizon V500), PercP and conjugates thereof. The conjugates may be PE-Cy5.5, PerCP-Cy5.5, PE-Cy7, and/or APC-H7.

The cells may be fixed and permeabilized with any method and reagent known in the art. For example, the cells may be permeabilized using a Cytofix/Cytoperm Kit (BD Biosciences).

The anti-CD8 antibody is preferably obtained from clone SK1 (sold by BD Biosciences, San Diego, Ca). The anti-CD27 antibody is preferably obtained from clone M-T271 (sold by BD Biosciences, San Diego, Ca). The anti-IFN-γ antibody is preferably obtained from clone 25723.1 1 (sold by BD Biosciences, San Diego, Ca). The anti-CCR7 antibody is preferably obtained from clone 3D12 (sold by BD Biosciences, San Diego, Ca). The anti-GM-CSF antibody is preferably obtained from clone MP1 -22E9 (sold by BD Biosciences, San Diego, Ca). The anti- CD3 antibody is preferably obtained from clone SK7 (sold by BD Biosciences, San Diego, Ca). The anti-CD38 antibody is preferably obtained from clone HIT2 (sold by BD Biosciences, San Diego, Ca). The anti-TNF-α antibody is preferably obtained from clone MAb1 1 (sold by BD Biosciences, San Diego, Ca). The anti-CD19 antibody is preferably obtained from clone SJ25C1 (sold by BD Biosciences, San Diego, Ca). The anti-CD45RO antibody is preferably obtained from clone UCHL1 (sold by BD Biosciences, San Diego, Ca). The anti-CD56 antibody is preferably obtained from clone NCAM16.2 (sold by BD Biosciences, San Diego, Ca). The anti- CD4 antibody is preferably obtained from clone RPA-T4 (sold by BD Biosciences, San Diego, Ca).

The kits disclosed herein are based on the predictive power of the method of the present invention. Reference values indicative of a responsive patient can be established using the method disclosed in the present application and the data disclosed in the Examples. In a preferred embodiment, a reference value which is indicative of a non-responsive patient (ODA) and/or a reference value which is indicative for a responsive patient (NEDA) may be provided with the kit. With the help of the kit, the quantity and/or percentages of each of the cell types can be calculated.

Further, it is to be interpreted that any embodiment of the kit can be used in the method of the present invention. Thus, subject matter related to the kit is also related to the method of the present invention. For example, the antibodies disclosed in the section entitled "Kit' can also be applied in the methods described in the section entitled "Method" of the present document.

In a further aspect, the kit according to any one of the aforementioned kit embodiments may be used in a method according to any one of the method embodiments described herein.

This is the first time that the lymphocyte profile of MS patients has been correlated to a particular MS patient subgroup which respond swell to DMF treatment. By determining this correlation, we can now use this information to predict whether a patient will respond well to a DMF treatment. Also, we can monitor the efficacy of the treatment over time. By being able to more quickly determine whether the treatment is likely to be effective or not, one can change the treatment for none-responders sooner to an alternative therapy which might be more effective.

The following examples merely illustrate the present invention.

Examples

Patient Demographic and clinical data

This study was approved by the ethics committee of Ramon y Cajal University Hospital, Madrid. Each patient signed a written consent before entry. A code was assigned to every patient to warrant confidentiality of collected data. We studied 64 patients diagnosed with RRMS who consecutively initiated treatment with DMF at the MS unit of Ramon y Cajal University Hospital. 65 RRMS patients (48 females and 17 males) were included in this study. However, only 64 of them were treated with DMF during at least one year, since a female patient abandoned treatment due to gastrointestinal side effects. Age at disease onset was 29.4 ± 1.3 years (Mean ± standard error); and disease duration at DMF initiation was 12.7 ± 1 .0 years. EDSS at this point was 2.8 ±0.2 and the annualized relapse rate in the previous two years 0.8 ± 0.1 . Eleven patients were treatment naive, 39 received previously first line drugs (lnterferon-β or Glatiramer acetate) and 15 second line drugs (Natalizumab, Fingolimod) or Azathioprine. Twenty-one patients were previously treated with only one disease modifying drug; 23 with two and 10 with three or more. Patients were classified according to their clinical and MRI activity after one year of follow-up. Those showing no evidence of disease activity in terms of absence of relapses, progression in the EDSS score or MRI activity were considered as optimal responders (NEDA). To have at least one relapse, or increase of at least one point in the EDSS score confirmed in two consecutive visits, or presence of new T2 lesions or gadolinium enhanced lesions was considered evidence of ongoing disease activity (ODA). After a year on DMF treatment, 41 patients showed NEDA and 23 ODA (6 patients suffered a relapse during follow-up, 1 1 had new lesions in the follow-up MRI study; 5 experienced disability worsening and the remaining one suffered a relapse and disability worsening). Baseline clinical and demographic data of patients showing NEDA or ODA are shown in Table 1 . No differences were found between the two groups of patients.

Table 1 : Clinical and demographic data of patients included in the study

NEDA (n=41) ODA (n=23) P

Age at disease onset (years) 30.7 ± 1.6 27.4 ^ 2.0 NS

(M±SE)

Sex (male/feinale) 10/31 7/16 NS

Age at baseline (years) (M±SE) 43.4 ± 1.2 39.9 = 2.7 NS

Disease duration (years) (M±SE) 12.6 ± 1.1 12.7 = 1.9 NS

EDSS score at baseline (M±SE) 2,8 ± 03 2.6 ± 0.3 NS

Relapses in the 2 years before DMF 0.7 ± 0.1 1.0 ± 0.2 NS treatment (M±SE)

Previous treatments 7 N: 25 FL; 9 SL 4 N: 14 FL: 5 SL NS

Number of previous treatments 7(N); 14(1); 14(2):6(>3) ^/ f(N); 7(1): 9(2): 3(>3) NS

Patient Follow-up

Patients were examined every three months, with additional visits in case of relapses. Relapses were defined as a worsening of neurological impairment or an appearance of a new symptom or abnormality attributable to MS, lasting at least 24 hours, and preceded by stability of at least one month¹⁷. EDSS score was assessed at each visit. A MRI imaging of the brain was performed within a month before treatment initiation and one year after starting DMF in 1.5 tesla scanners with standard head coils (Philips Gyroscan NT, Eindhoven, Netherlands). Slice thickness of 5 mm were acquired to obtain contiguous axial sections that covered the entire brain. The following sequences were performed: T1 weighted imaging, T1 weighted imaging with gadolinium enhancement, axial FLAIR T2, axial T2 weighted imaging and axial proton density T2 weighted imaging.

Samples

Samples were collected immediately before treatment with DMF and after six months of treatment. Heparinized whole blood was collected and peripheral blood mononuclear cells (PBMC) obtained within 2 hours by Ficoll density gradient centrifugation (Fresenius Kabi, Norway) and cryopreserved in aliquots of 5-6x10⁶ cells until studied. Monoclonal antibodies

The following monoclonal antibodies were used in the study: CD8-FITC, CD27-FITC, lnterferon(IFN)-gamma-FITC, CD24-PE, CD197-PE (CCR7-PE), Granulocyte/macrophage- colony stimulating factor (GM-CSF)-PE, CD3-PerCP, CD38-PE-Cy5.5, TNF-alpha-PerCP- Cy5.5, CD19-PE-Cy7, CD25-PE-Cy7, CD45RO-APC, CD56-APC, CD4-APC-H7, CD8-APC-H7, CD3-BV421 , CD127-BV421 , CD45-V500 (all from BD Biosciences, San Diego, CA); interleukin (IL)-10-PE (Biolegend, San Diego, CA) and IL-17-APC (R&D Systems, Minneapolis, MN).

Statistical analysis

Statistical analyses were made using GraphPad Prism 6.0 software (GraphPad Prism Inc, La Jolla, CA). Wilcoxon matched pair tests were used to assess differences between basal and six months after treatment results; Mann-Whitney test was used to compare results in NEDA versus ODA group; Correlations were assessed by a Spearman test, p values below 0.05 were considered as significant. Labelling of surface antigens

Aliquots of 10⁶ PBMC were resuspended in RPMI 1640 medium (Thermofisher Scientific, Waltham, MA), stained with the appropriate amounts of monoclonal antibodies during 30 minutes at 4°C in the dark, washed twice with PBS, resuspended in 300 μΙ of PBS and analyzed.

In vitro stimulation and intracellular cytokine staining

Aliquots of 10⁶ PBMC were resuspended in 1 ml of Complete Medium with 50 ng/ml Phorbol 12- myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO) and 750 ng/ml lonomycin (Sigma- Aldrich), in presence of 2 μg/ml Brefeldin A (GolgiPlug, BD Biosciences) and 2.1 μΜ Monensin (Golgi Stop, BD Biosciences) in polypropylene tubes, and incubated for 4 hours at 37°C in 5% C0₂. To identify IL-10 producing B cells, PBMC were preincubated with 3 μg/ml of CpG oligonucleotide (InvivoGen, San Diego, CA) during 20 h at 37°C with 5% C0₂ prior to stimulation. After incubation, PBMC were washed with PBS, resuspended in residual volume and stained for 30 minutes at 4°C in the dark with the appropriate amounts of monoclonal antibodies recognizing the surface antigens. Then, cells were washed with PBS, fixed and permeabilized for 20 min at 4°C in the dark with Cytofix/Cytoperm Kit (BD Biosciences), washed twice with Perm/Wash solution (BD Biosciences) and stained intracellular^ 30 min at 4°C in the dark with monoclonal antibodies recognizing the following cytokines: IL-10, IFN-gamma, GM-CSF-PE, TNF-alpha and IL-17. After two washes, PBMC were analyzed in a FACSCanto II flow cytometer (BD Biosciences). Flow cytometry

Cells were always analyzed within a maximum period of 1 hour after staining. Mean autofluorescence values were set using appropriate negative isotype controls. Data analysis was performed using FACSDiva Software V.8.0 (BD Biosciences). A gate including lymphocytes and monocytes and excluding debris and apoptotic cells was established; a minimum amount of 30.000 events were analyzed. According to the differential expression of several antigens, CD4+ and CD8+T cells were classified as: naive (CCR7+ CD45RO-); central memory (CM) (CCR7+ CD45RO+); effector memory (EM) (CCR7- CD45RO+); terminally differentiated (TD) (CCR7- CD45RO-). Regulatory CD4 T cells (Treg) were defined as CD3+ CD4+ CD25hi CD127low. B cells were classified as: memory (CD19+ CD27dim CD38dim), plasmablasts (CD19+ CD27hi CD38hi); and regulatory (Breg) (CD19+ CD27- CD24hi CD38hi or CD19+ IL-10+); we also explored natural killer (NK) cells (CD56dim CD3-), natural killer T (NKT) cells (CD56dim CD3+), and CD56bright NK cells (CD3- CD56bright). For intracellular cytokine staining, non-stimulated PBMC and appropriate isotype controls were used. Example 1 : Effector and regulatory subsets

We first studied different immune cell subsets in blood mononuclear cells obtained before and six months after treatment initiation in the whole group of patients (n=64). We found significant changes in effector, memory and regulatory cells subsets. The percentages of total CD8 T cells, CD4+ and CD8+ CM cells, EM and TD T cells, NKT cells and memory B cells decreased at six months of treatment with DMF, while those of CD4+ and CD8+ naive T cells, and CD56bright cells increased. Results are shown in table 2.

Table 2: DMF induced changes in lymphocyte blood subsets

We next explored the same subsets in NEDA and ODA patients. No difference in any particular subpopulation was observed between both groups prior to treatment onset (data not shown).When we studied changes between basal and six month samples (table 2), both groups showed similar increases in naive CD4+ and CD8+ T cells and similar decreases in EM CD4+ T cells, totalCD8+ T cells and in EM and TD CD8+ T cells during treatment. However, NEDA patients showed a distinct profile of other lymphoid subsets. They showed a clear decrease in CM CD4+ and CD8+T cells (p = 0.0004 and p=0.0008 respectively) (Representative examples shown in figure 1 ), while no significant changes were appreciated in these subsets in ODA group. In the same line, NKT cells and memory B cells experienced a higher decrease and CD56bright cells a higher increase in NEDA than in ODA patients. Example 2: Intracellular cytokine production

We next studied intracellular cytokine production in T and B lymphocytes. We found a significant decrease in the percentage of CD4+ and CD8+ IFN-gamma+ T cells, CD8+ TNF- alpha+ T cells, CD8+ GM-CSF+ T cells and CD19+ TNF-alpha+ B cells after six months of DMF treatment in the whole group of patients (table 3). These differences were restricted in most cases to NEDA group. The unique subset that decreased significantly in ODA patients was CD8+ T cells producing TNF-a+, although differences were lower than in NEDA group

Table 3: DMF induced changes in cytokine production by blood lymphocytes

Total Patients (ii = 64) NEDA (ii=41) ODA (ii=23)

Variable Basal 6 Mo P Basal 6 Mo P Basal 6 Mo P

(M+SE) (M+SE) (M+SE) (M+SE) (M±SE) (M+SE)

CD4+IFN-y+ 4.5±0.3 2.8*0.3 < 0.0001 4.5+0.4 2.3+0.4 < 0.0001 4.4+0.5 3.3+0.4 NS

CD4+ INF-0+ 18.6±1.2 19.6±1.1 NS 19.1+1.5 18.5+1.3 NS 17.5+1.9 21.3+2.1 NS

CD4+IL-17+ 0.5±0.05 0.6+0,06 NS 0.5+0.05 0.5+0.06 NS 0.6+0.11 0.8=0.13 NS

CD4+GM-CSF⁺ 3.9+0.3 4.0+0.3 NS 4.1+0.4 4.0+0.3 NS 3.5+0.4 4.1±0.5 NS

CD4+ IL-10+ 0.3±0.03 0.3±0.02 NS 0,3+0.04 0,3+0.03 NS 0.3+0.04 0.3+0.04 NS

CD8+ IFN-7+ 5.0±0.4 3.3+0.4 < 0.0001 4.8+0.5 3.2+0.6 < 0.0001 5.0±0.8 3.4+0.6 NS

CD8+TNF-a- 6.7+0.5 4.7+0.5 < 0.0001 6.3+0.5 4.6+0.8 < 0.0001 7.1±0.9 5.0±0.6 0.02

CD8+ IL-17+ 0.2±0.03 0.2+0,03 NS 0.2+0.04 0.3+0.05 NS 0.2+0.05 0.2-0.05 NS

CD8+ GM-CSF+ 1.6+0.1 1.2+0.12 0.0028 1.5+0.2 1.2+0.1 0.01 1.6+0.2 1.2*0.2 NS

CD8+ IL-10+ 0,2±0.04 0.2+0.02 NS 0.2+0,04 0,2+0.03 NS 0.3+0.06 0.1+O.Oi NS

CD19+TNF0C+ 2.8+0,3 2.1+0.3 0.0062 2.6±0.3 1.6+0.3 < 0,0001 2.9+0.5 2.9+0.5 NS

CD 19+GM-CSF+ 0.6±0.05 0.6±0.06 NS 0.7+0.06 0,7+0.09 NS 0.5±0.09 0.5±0.09 NS

CD1 +IL-10+ 0.2±0,02 0.2+0.02 NS 0.2+0.02 0.2+0.03 NS 0.2*0.03 0.2+0.03 NS

Thus, the decrease in intracellular cytokine production induced by DMF is mainly restricted to NEDA group, which strongly suggests that this mechanism is associated with optimal response to this drug. A representative example of the changes in cytokine producing subsets in NEDA patients is shown in figure 2.

Example 3: Association between regulatory and effector populations

Since we observed a considerable increase in NK CD56bright and a clear decrease in different effector cells during DMF treatment, we explored the putative correlation between these regulatory cells and the effector subsets after 6 months of treatment. The clearest results were obtained with some cytokine producing subsets. There was a negative correlation within the percentage of CD56bright cells and those of CD8+ T cells producing TNF-a (r= -0.48, p<0.0001 ) and IFN-γ (r= -0.41 , p=0.0012) and to a lesser extent with CD4+ T cells producing IFN-γ (r= - 0.28, p=0.028). These data reveal a potential mechanism of action for DMF by upregulating CD56bright cells and inducing a secondary inhibition of pro-inflammatory effector cells. Results are shown in table 4.

Table 4: Correlation between the percentages of lymphocytes producing proinflammatory cytokines and of CD56bright cells at six months of DMF treatment

Effector population r P

CD4+ IFN-ganima+ 412815 0.0280

CD4+ TNF-gamma+ -0.0064 0.96

CD4+ IL- 17+ 410377 0.77

CD4+ GM-CSF+ 0.2483 0. 10

CD8+ IFN-gamma+ -0.4080 0.0012

CD8+ TNF-alpha+ -0.4824 < 0.0001

C D8+ IL- 17+ -0.0832 0.52

CD8+ GM-CSF+ 0.127 1 0.41

CD 1 + TNF-alpha+ -0.2296 0.08

CD 19+ GM-CSF+ 0.0450 0.78

References

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Claims

1 . A method for monitoring and/or predicting the efficacy of a dimethyl fumarate treatment in a patient who suffers from Multiple Sclerosis, comprising the steps of:

(a) isolating peripheral blood mononuclear cells from isolated blood samples obtained before and after dimethyl fumarate treatment; and

(b) quantifying the amount of cells of one or more peripheral blood mononuclear cell subset in the before and after sample.

2. The method according to claim 1 , wherein the cells quantified in step (b) are CD4+ central memory cells, CD8+ T cells, CD8+ central memory cells, natural killer T cells, memory B cells, CD56bright natural killer cells, CD4+IFN-y+ T cells, CD8+IFN-y+ T cells, CD8+TNF-a+ T cells, CD8+GM-CSF+ T cells and/or CD19+TNF-a+ B cells.

3. The method according to any one of claims 1 -2 which further comprises the step of: (c) assigning those patients who exhibit a:

(i) decrease in the amount of CD4+ central memory cells, CD8+ T cells, CD8+ central memory cells, natural killer T cells and/or memory B cells;

(ii) an increase in the amount of CD56bright natural killer cells; and/or

(iii) a decrease in CD4+IFN-y+ T cells, CD8+IFN-y+ T cells, CD8+TNF-a+ T cells, CD8+GM- CSF+ T cells, and/or CD19+TNF-Q+ B cells

after dimethyl fumarate treatment to the group of patients who have a higher probability of responding positively and/or optimally to a dimethyl fumarate treatment.

4. The method according to any one of claims 1-3, wherein the Multiple Sclerosis is relapsing remitting Multiple Sclerosis.

5. The method according to any one of the preceding claims, wherein the isolated blood samples were obtained before and after a dimethyl fumarate treatment which lasted for at least 0, 1 , 2, 3, 4 or 5 months, preferably at least 6 months.

6. The method according to any one of the previous claims, wherein the amount of CD56bright natural killer cells is correlated with the amount of CD4+IFN-y+ T cells, CD8+IFN-y+ T cells and/or CD8+TNF-a+ T cells and patients who exhibit a negative correlation between CD56bright natural killer cells and CD4+IFN-y+ T cells, CD8+IFN-y+ T cells and/or CD8+TNF- a+ T cells after dimethyl fumarate treatment are assigned to the group of patients who have a higher probability of responding positively and/or optimally to a dimethyl fumarate treatment.

7. The method according to any one of the previous claims, wherein the peripheral blood mononuclear cells are isolated from the isolated blood samples by using centrifugation, preferably Ficoll density gradient centrifugation.

8. The method according to any one of the previous claims, wherein the amount of cells are quantified using flow cytometry.

9. The method according to claim 7, wherein the isolated peripheral blood mononuclear cells are stained using antibodies recognizing surface antigens.

10. The method according to any one of claims 7-8, wherein the peripheral blood mononuclear cells are permeabilized and stained intracellularly with antibodies recognizing cytokines.

1 1 . A kit comprising:

(i) antibodies for identifying CD4+ central memory cells, CD8+ T cells, CD8+ central memory cells, natural killer T cells, memory B cells, CD56bright natural killer cells, CD4+IFN-y+ T cells, CD8+IFN-Y+ T cells, CD8+TNF-Q+ T cells, CD8+GM-CSF+ T cells and/or CD19+TNF-Q+ B cells.

12. The kit according to claim 1 1 , wherein the kit comprises:

(i-a) antibodies for identifying CD56bright natural killer cells and CD4+IFN-y+ T cells;

(i-b) antibodies for identifying CD56bright natural killer cells and CD8+IFN-y+ T cells; and/or

(i-c) antibodies for identifying CD56bright natural killer cells and CD8+TNF-Q+ T cells.

13. The kit according to claims 1 1 or 12, wherein

the antibodies used to ident fy CD4+ central memory cells bind to CD4, CCR7 and CD45RO; the antibodies used to ident fy CD8+ T cells bind to CD8;

the antibodies used to ident fy CD8+ central memory cells bind to CD8, CCR7 and CD45RO; the antibodies used to ident fy natural killer T cells bind to CD56 and CD3;

the antibodies used to ident fy memory B cells bind to CD19, CD27 and CD38;

the antibodies used to ident fy CD56bright natural killer cells bind to CD56 and CD3;

the antibodies used to ident fy CD4+IFN-Y+ T cells bind to CD4 and IFN-γ;

the antibodies used to ident fy CD8+IFN-Y+ T cells bind to CD8 and IFN-γ;

the antibodies used to ident fy CD8+TNF-Q+ T cells bind to CD8 and TNF-a;

the antibodies used to ident fy CD8+GM-CSF+ T cells bind to CD8 and GM-CSF; and/or the antibodies used to ident fy CD19+TNF-Q+ B cells bind to CD19 and TNF-a.

14. The kit according to claim 13, wherein the antibodies are labeled.

15. The kit according to claim 14, wherein

the labeled antibodies used to identify CD4+ central memory cells are anti-CD4-APC-H7, anti- CCR7-PE and anti-CD45RO-APC;

the labeled antibody used to identify CD8+ T cells is anti-CD8-APC-H7 and/or anti-CD8-FITC; the labeled antibodies used to identify CD8+ central memory cells are anti-CD8-APC-H7 and/or anti-CD8-FITC, anti-CCR7-PE and anti-CD45RO-APC;

the labeled antibodies used to identify natural killer T cells are anti-CD56-APC, and anti-CD3- BV421 and/or anti-CD3-PerCP;

the labeled antibodies used to identify memory B cells are anti-CD19-PE-Cy7, anti-CD27-FITC and anti-CD38-PE-Cy5.5;

the labeled antibodies used to identify CD56bright natural killer cells are anti-CD56-APC, and anti-CD3-BV421 and/or anti-CD3-PerCP;

the labeled antibodies used to identify CD4+IFN-V+ T cells are anti-CD4-APC-H7 and anti-IFN- Y-FITC;

the labeled antibodies used to identify CD8+IFN-y+ T cells are anti-CD8-APC-H7 and/or anti- CD8-FITC, and anti-IFN-v-FITC;

the labeled antibodies used to identify CD8+TNF-a+ T cells are anti-CD8-APC-H7 and/or anti- CD8-FITC, and anti-TNF-a-PerCP-Cy5.5;

the labeled antibodies used to identify CD8+GM-CSF+ T cells are anti-CD8-APC-H7 and/or anti-CD8-FITC, and anti-GM-CSF-PE; and/or

the labeled antibodies used to identify CD19+TNF-a+ B cells are anti-CD19-PE-Cy7 and anti- TNF-a-PerCP-Cy5.5.

16. The kit according to claim 1 1 which comprises antibodies used to identify CD4+ central memory cells, wherein the kit comprises:

(i) a tube or recipient comprising monoclonal antibodies anti-CD4, anti-CCR7 and anti-CD45RO, or several tubes or recipients comprising separately or in combination each of these antibodies, and

(ii) optionally a washing or buffer solution.

17. The kit according to claim 16 wherein the antibodies are labeled and the anti-CD4 antibody is anti-CD4-APC-H7, anti-CCR7 is anti-CCR7-PE, and anti-CD45RO is anti-CD45RO- APC.

18. The kit according to claim 1 1 which comprises antibodies used to identify CD8+ T cells, wherein the kit comprises: (i) a tube or recipient comprising monoclonal antibodies anti-CD8, and

(ii) optionally a washing or buffer solution.

19. The kit according to claim 18 wherein the antibodies are labeled and the anti-CD8 antibody is anti-CD8-APC-H7 and/or anti-CD8-FITC.

20. The kit according to claim 1 1 which comprises antibodies used to identify CD8+ central memory cells, wherein the kit comprises:

(i) a tube or recipient comprising monoclonal antibodies anti-CD8, anti-CCR7 and anti-CD45RO, or several tubes or recipients comprising separately or in combination each of these antibodies, and

(ii) optionally a washing or buffer solution.

21 . The kit according to claim 20 wherein the antibodies are labeled and the anti-CD8 antibody is anti-CD8-APC-H7 and/or anti-CD8-FITC, anti-CCR7 is anti-CCR7-PE, and anti- CD45RO is anti-CD45RO-APC.

22. The kit according to claim 1 1 which comprises antibodies used to identify natural killer T cells, wherein the kit comprises:

(i) a tube or recipient comprising monoclonal antibodies anti-CD56 and anti-CD3, or several tubes or recipients comprising separately or in combination each of these antibodies, and

(ii) optionally a washing or buffer solution.

23. The kit according to claim 22 wherein the antibodies are labeled and the anti-CD56 antibody is anti-CD56-APC, and anti-CD3 is anti-CD3-BV421 and/or anti-CD3-PerCP.

24. The kit according to claim 1 1 which comprises antibodies used to identify memory B cells, wherein the kit comprises:

(i) a tube or recipient comprising monoclonal antibodies anti-CD19, anti-CD27 and anti-CD38, or several tubes or recipients comprising separately or in combination each of these antibodies, and

(ii) optionally a washing or buffer solution.

25. The kit according to claim 24 wherein the antibodies are labeled and the anti-CD19 antibody is anti-CD19-PE-Cy7, anti-CD27 is anti-CD27-FITC, and anti-CD38 is anti-CD38-PE- Cy5.5.

26. The kit according to claim 1 1 which comprises antibodies used to identify CD56bright natural killer cells, wherein the kit comprises:

(ii) optionally a washing or buffer solution.

27. The kit according to claim 26 wherein the antibodies are labeled and the anti-CD56 antibody is anti-CD56-APC, and anti-CD3 is anti-CD3-BV421 and/or anti-CD3-PerCP.

28. The kit according to claim 1 1 which comprises antibodies used to identify CD4+IFN-y+ T cells, wherein the kit comprises:

(i) a tube or recipient comprising monoclonal antibodies anti-CD4 and anti-IFN-γ, or several tubes or recipients comprising separately or in combination each of these antibodies,

(ii) optionally a washing or buffer solution, and

(iii) optionally means for the fixation and permeabilization of cells and/or a buffer to wash the cells and to dilute the anti-cytokine antibodies for staining.

29. The kit according to claim 28 wherein the antibodies are labeled and the anti-CD4 antibody is anti-CD4-APC-H7, and anti-IFN-γ is anti-IFN-v-FITC.

30. The kit according to claim 1 1 which comprises antibodies used to identify CD8+IFN-y+ T cells, wherein the kit comprises:

(i) a tube or recipient comprising monoclonal antibodies anti-CD8 and anti-IFN-γ, or several tubes or recipients comprising separately or in combination each of these antibodies,

(ii) optionally a washing or buffer solution, and

31 . The kit according to claim 30 wherein the antibodies are labeled and the anti-CD8 antibody is anti-CD8-APC-H7 and/or anti-CD8-FITC, and anti-IFN-γ is anti-IFN-Y-FITC.

32. The kit according to claim 1 1 which comprises antibodies used to identify CD8+TNF-a+ T cells, wherein the kit comprises:

(i) a tube or recipient comprising monoclonal antibodies anti-CD8 and anti-TNF-a, or several tubes or recipients comprising separately or in combination each of these antibodies,

(ii) optionally a washing or buffer solution, and

33. The kit according to claim 32 wherein the antibodies are labeled and the anti-CD8 antibody is anti-CD8-APC-H7 and/or anti-CD8-FITC, and anti-TNF-a is anti-TNF-a-PerCP- Cy5.5.

34. The kit according to claim 1 1 which comprises antibodies used to identify CD8+GM- CSF+ T cells, wherein the kit comprises:

(i) a tube or recipient comprising monoclonal antibodies anti-CD8 and anti-GM-CSF, or several tubes or recipients comprising separately or in combination each of these antibodies,

(ii) optionally a washing or buffer solution, and

35. The kit according to claim 34 wherein the antibodies are labeled and the anti-CD8 antibody is anti-CD8-APC-H7 and/or anti-CD8-FITC, and anti-GM-CSF is anti-GM-CSF-PE.

36. The kit according to claim 1 1 which comprises antibodies used to identify CD19+TNF-a+ B cells, wherein the kit comprises:

(i) a tube or recipient comprising monoclonal antibodies anti-CD19 and anti-TNF-a, or several tubes or recipients comprising separately or in combination each of these antibodies,

(ii) optionally a washing or buffer solution, and

37. The kit according to claim 36 wherein the antibodies are labeled and the anti-CD19 antibody is anti-CD19-PE-Cy7, and anti-TNF-α is anti-TNF-a-PerCP-Cy5.5.

38. The kit according to claim 12 which comprises antibodies used to identify CD56bright natural killer cells and antibodies used to identify CD4+IFN-y+ T cells, wherein the kit comprises the components of claims 26 or 27, and 28 or 29.

39. The kit according to claim 12 which comprises antibodies used to identify CD56bright natural killer cells and antibodies used to identify CD8+IFN-y+ T cells, wherein the kit comprises the components of claims 26 or 27, and 30 or 31.

40. The kit according to claim 12 which comprises antibodies used to identify CD56bright natural killer cells and antibodies used to identify CD8+TNF-a+ T cells, wherein the kit comprises the components of claims 26 or 27, and 32 or 33.

41 . The kit according to any one of claims 1 1 -40, wherein the kit further comprises dimethyl fumarate.

42. Use of the kit according to any one of claims 1 1 -41 for the method of any one of claims 1 -10.