Methods for predicting active disease in a subject suffering from multiple sclerosis

Abstract

Multiple sclerosis (MS) is a neuroinflammatory disease driven by B- and T-cells. The role of follicular regulatory T cells (Tfr) in MS remains ill-defined. Here, we found the clinical activity of MS patients to follow the frequency of circulating Tfr, suggesting that Tfr promote clinical relapse. Furthermore, Tfr-deficient mice underwent less severe experimental autoimmune encephalomyelitis than wild-type mice, which correlated with a decrease in B- cells infiltrating the central nervous system (CNS). Mechanistically, Tfr deficiency led to the trapping of B-cells in germinal centers of second lymphoid organs through B-cell overexpression of the sphingosine-1-phosphate receptor 2 (S1PR2). Tfr conversely promoted encephalomyelitis by downregulating S1PR2, which allowed B-cells to egress from germinal centers and migrate to the CNS, where they promoted cytokine production by encephalitogenic T-cells. Together, these findings demonstrate that Tfr contribute to autoimmune encephalomyelitis, and that Tfr frequency in blood reflects MS activity. Accordingly, the present invention relates to a method for predicting active disease in a subject suffering from multiple sclerosis, said method comprising the step of determining the frequency of Follicular Regulatory T (Tfr) cells in a population of cells in a sample obtained from the subject.

Description

[0001] METHODS FOR PREDICTING ACTIVE DISEASE IN A SUBJECT SUFFERING FROM MULTIPLE SCLEROSIS

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine and relates in particular to autoimmune diseases.

[0004] BACKGROUND OF THE INVENTION:

[0005] Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) that is characterized by inflammation, demyelination and axonal damage. It is likely induced by an autoimmune reaction, which attacks and damages the myelin sheath as well as the axons and neurons, leading to various neurological symptoms1. While it is commonly accepted that encephalitogenic T-cells, through pro-inflammatory cytokines, play a major role in this disease2,3, recent studies have demonstrated that B-cells play also a significant role in MS pathogenesis1,4,5, mainly through B- and T-cell collaboration6’7. B-cells can contribute to the disease process through multiple mechanisms: in situ reactivation of encephalitogenic T-cells through antigen presentation8, pro-inflammatory cytokine production9 1formation of ectopic lymphoid follicles within the meninges12, and, to a lesser extent, autoantibody production against components of myelin13’14. Due to the involvement of B-cells in the pathogenesis of MS, therapies inducing B-cell depletion are used for the treatment of relapsing remitting MS (RRMS), yielding remarkable outcomes. Additionally, some beneficial effects have been observed in active primary progressive MS15 l7. Follicular regulatory T-cells (Tfr) are a subset of Foxp3+regulatory T-cells (Tregs) that play a critical role in regulating the immune response within germinal centers (GC) where B-cell responses undergo affinity maturation processes18,19. There, B-cells are confined in GC through the expression of sphingosine- 1 -phosphate receptor 2 (S1PR2), a member of the family of sphingosine- 1 -phosphate (SIP)20,21. These cells undergo activation-induced cytidine deaminase (AID)-mediated somatic hypermutation of immunoglobulin and the ones with high-affinity are selected and released as memory B-cells and long-lived plasma cells22. Tfr exhibit a phenotypical resemblance to T follicular helper cells (Tfh) in numerous aspects, including the expression of the chemokine receptor CXCR5 and the transcription factor Bcl623 25, which allow their migration into GC. Tfr control GC size, duration and output, particularly by suppressing Tfh and GC-B cells18,26 30. Being regulatory T-cells, Tfr are involved in the maintenance of immune tolerance to self-antigens, and theirdysfunction or loss can lead to the development of autoimmune responses30. In the context of autoimmunity, Tfr have been shown to control autoantibody production, by limiting the numbers of Tfh and / or inhibiting plasma cell differentiation in mice29,30. Furthermore, in patients with systemic autoimmune diseases such as systemic lupus erythematosus31, rheumatoid arthritis and Sjogren’s syndrome32, circulating Tfr numbers are decreased and these cells display some functional defects, both thought to contribute to the dysregulated autoantibody responses seen in these diseases. Given their impact on immune tolerance, Tfr dysfunction could also play a crucial role in organ-specific autoimmune diseases, such as MS. As of now, it was shown that circulating Tfr in MS patients were decreased in numbers33,34while other studies observed no differences in the frequency of circulating Tfr35,36. Hence, a deeper understanding of Tfr functions in MS may provide important insights into the pathogenesis and potential therapeutic targets for this disease.

[0006] SUMMARY OF THE INVENTION:

[0007] The present invention is defined by the claims. In particular, the present invention relates to a method for predicting active disease in a subject suffering from multiple sclerosis, said method comprising the step of determining the frequency of Follicular Regulatory T (Tfr) cells in a population of cells in a sample obtained from the subject.

[0008] DETAILED DESCRIPTION OF THE INVENTION:

[0009] In this context, the Inventors found that the circulating Tfr frequency was lower in RRMS patients in remission as compared to RRMS patients in relapse. Moreover, RRMS patients treated with Natalizumab, a humanized monoclonal antibody targeting the cell adhesion molecule a4-integrin, exhibited a decrease in the circulating Tfr frequency in parallel to the treatment response and the decrease in clinical activity. To confirm that these Tfr promote clinical activity, they used a B cell-dependent pre-clinical mouse model of MS, an experimental autoimmune encephalomyelitis (EAE) induced by immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG)37. They found that absence of Tfr led to a less severe pathology. They demonstrated that, in absence of Tfr, fewer B-cells migrate to the brain at the peak of the disease, which resulted in a decrease of pro-inflammatory cytokine production by brain-infiltrating T-cells. Furthermore, they showed that B-cells were not migrating to the brain since draining lymph node (dLN) B-cells were expressing higher levels of S1PR2 in absence of Tfr, which trapped them in GC from dLN. Notably, treatment of EAE induced Tfr-deficient mice with a S1PR2 antagonist allowed brain infiltration by B-cells, pro-inflammatorycytokine production by brain T-cells and restored the severity of EAE. Overall, they thus demonstrated that Tfr unexpectedly contribute to autoimmune encephalomyelitis by promoting the egress of B-cells from GC.

[0010] As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine or a primate. In a preferred embodiment, the subject is a human. In some embodiments, the subject is asymptomatic. In some embodiments, the subject is remitting. In some embodiments, the subject is relapsing. In some embodiments, the subject suffers from relapsing-remitting multiple sclerosis. In some embodiments, the subject suffers from clinically isolated syndrome (i.e. a single episode of neurological symptoms caused by inflammation or demyelination in the central nervous system, progressing or not to MS), relapsing-remitting multiple sclerosis (i.e. characterized by clearly defined relapses - flare-ups or attacks - of symptoms followed by periods of partial or complete recovery - remission), primary progressive multiple sclerosis (i.e. characterized by a gradual worsening of neurological function from the beginning, without distinct relapse or remissions) or secondary progressive multiple sclerosis (i.e. a stage of MS that follow RRMS, where the disease steadily worsens over time, with or without relapse). In some embodiments the subject suffers from active multiple sclerosis.

[0011] As used herein, the term “multiple sclerosis” or “MS” refers to a central nervous system autoimmune condition. MS is a demyelinating disease resulting in damage to the insulating covers of nerve cells in the brain and spinal cord. MS disrupts the nervous system’s ability to transmit signals, leading to a range of symptoms including physical, mental and / or psychiatric problems, such as double vision, vision loss, eye pain, memory trouble, concentration trouble, mood change, depression, anxiety, muscle weakness, muscle spasms, muscle stiffness, loss of bowel or bladder control, spasticity, fatigue, tremor, dizziness, clumsiness, loss of sensation or loss of coordination. As example, diagnosis can be based on clinical evidence or MRI evidence of at least two characteristic neurological lesions that are separated in time and space (location in the central nervous system). Diagnostic testing may also include blood and urine tests, magnetic resonance imaging test, optical coherence tomography and lumbar puncture.

[0012] As used herein, the term “active disease” or “active multiple sclerosis” refers to a phase of the disease characterized by ongoing disease activity, which can be detected through clinical symptoms, imaging or other diagnostic methods. The term includes both evidence of activity(i.e. new or enlarging lesions) and clinical relapse (i.e. episode of new or worsening neurological symptoms after a recovery period).

[0013] As used herein, the term “Follicular Regulatory T cells” or “Tfr” refers to a specialized subset of CD4+ regulatory T cells (Tregs) that play a critical role in maintaining immune homeostasis within germinal centers (GCs). The phenotype of Tfr can vary depending on their activation state, tissue localization and functional role. In some embodiments, the Tfr cells are CD3+, CD4+, CD45RA’, CXCR5+, Foxp3+or CD1271ow CD25high or CD1271ow Foxp3+. In some embodiments, the Tfr cells are CD3+, CD4+, CD45RA', CXCR5+and / or Foxp3+.

[0014] Methods for predicting or diagnosing active disease

[0015] In a first aspect, the present invention relates to a method for predicting or diagnosing active disease in a subject suffering from multiple sclerosis, comprising the step of determining the frequency of Tfr cells in a population of cells in a sample obtained from the subject. In some embodiments, the present invention relates to a method for predicting or diagnosing active disease in a subject suffering from multiple sclerosis, comprising the step of determining the frequency of CD3+CD4+CD45RA CXCR5+Foxp3+cells in a population of cells in a sample obtained from the subject.

[0016] As used herein, the term “predicting active disease” includes detection of active disease before clinical symptoms, before it can be diagnosed by imaging or other diagnostic methods. The term includes predicting both evidence of activity (i.e. new or enlarging lesions) and clinical relapse (i.e. episode of new or worsening neurological symptoms after a recovery period).

[0017] As used herein, the term “sample” refers to any biological sample obtained for the purpose of evaluation in vitro or ex vivo, such as whole blood, serum, plasma, amniotic fluid, brain / spinal cord fluid, liquor, cerebrospinal fluid, sputum, throat and pharynx secretions and other mucous membrane secretions, synovial fluids, ascites, tear fluid, lymph fluid and urine sample. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a peripheral blood sample. In some embodiments, the sample is a sample of (i) purified blood leukocytes, (ii) peripheral blood mononuclear cells or PBMC, (iii) purified lymphocytes, (iv) purified T cells, (v) purified TCD4+cells or (vi) red blood cells lysis sample. In some embodiments, the sample comprises CD4+T cells. In some embodiments, the population of cells in the sample is a population of CD4+T cells.In some embodiments, the present invention relates to a method for predicting active disease in a subject suffering from multiple sclerosis, said method comprising the steps of:

[0018] - Determining the frequency of Tfir cells in a population of cells in a sample obtained from the subject; and

[0019] Concluding that the subject will suffer from active multiple sclerosis when the frequency of Tfr cells, is higher than a predetermined reference value.

[0020] In some embodiments, the present invention relates to a method for diagnosing active disease in a subject suffering from multiple sclerosis, said method comprising the steps of:

[0021] - Determining the frequency of Tfr cells in a population of cells in a sample obtained from the subject; and

[0022] Concluding that the subject suffers from active multiple sclerosis when the frequency of Tfr cells, is higher than a predetermined reference value.

[0023] In some embodiments, the present invention also relates to a method for predicting active disease in a subject suffering from multiple sclerosis, said method comprising the steps of:

[0024] Determining the frequency of CD3+CD4+CD45RACXCR5+Foxp3+cells in a population of cells in a sample obtained from the subject; and

[0025] Concluding that the subject will suffer from active multiple sclerosis when the frequency of CD3+CD4+CD45RA CXCR5+Foxp3+cells is higher than a predetermined reference value.

[0026] In some embodiments, the present invention also relates to a method for diagnosing active disease in a subject suffering from multiple sclerosis, said method comprising the steps of:

[0027] Determining the frequency of CD3+CD4+CD45RACXCR5+Foxp3+cells in a population of cells in a sample obtained from the subject; and

[0028] Concluding that the subject suffers from active multiple sclerosis when the frequency of CD3+CD4+CD45RACXCR5+Foxp3+cells is higher than a predetermined reference value.

[0029] CD3, CD4, CD45RA, CXCR5 and Foxp3 and others are considered below as biomarker of follicular regulatory T cells (“Tfr” cells). As used herein, the term “frequency of cells” refers to the proportion or number of specific cells present in a circulating biological sample (e.g. blood or bodily fluid) relative to a total cell population (e.g. CD4+ population) or a givenvolume (e.g. cells / mL). The frequency can be expressed in relative terms (e.g. percentage) or absolute terms (e.g number of cells per unit volume). In some embodiments, the frequency of Tfr cells, in particular CD3+CD4+CD45RACXCR5+Foxp3+cells, is calculated relative to total CD4+ population. In some embodiments, the frequency of the Tfr cells, in particular CD3+CD4+CD45RA CXCR5+Foxp3+cells, represents 1, 2, 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% of the total CD4+ cell population. The frequency may also be expressed as a ratio, such as a ratio of Tfr cells to Tfh cells (e.g. CD3+CD4+CD45RA CXCR5+Foxp3‘ cells). Typically, an increased ratio of Tfr cells to Tfh cells is indicative of an active multiple sclerosis.

[0030] In some embodiments, the frequency of the biomarkers is determined by a flow-cytometric method. As used herein, the term "flow cytometric method" refers to a technique for counting cells of interest, by suspending them in a stream of fluid and passing them through an electronic detection apparatus. Flow cytometric methods allow simultaneous multiparametric analysis of the physical and / or chemical parameters of up to thousands of events per second, such as fluorescent parameters. Modern flow cytometric instruments usually have multiple lasers and fluorescence detectors. In some embodiments, the frequency of the biomarkers is determined by the flow cytometric method described in material and methods section (see “flow cytometry analysis and cell sorting”). For cell surface staining, fresh PBMC from blood samples are used after Ficoll or Percoll purification or red blood cell lysis.

[0031] The cytometric systems of the present disclosure may include a number of additional components, such as data output devices, e.g., monitors, printers, and / or speakers, softwares (e.g. Flowjo, DIVA, CytoBank....), data input devices, e.g., interface ports, a mouse, a keyboard, etc., fluid handling components, power sources, etc. More particularly, the sample is contacted with a panel of antibodies specific for the specific markers of the population of cells of the interest. Such antibodies or antigen-binding fragments are available commercially from vendors such as R&D Systems, BD Biosciences, e- Biosciences, Biolegend, Proimmune and Miltenyi, or can be raised against these cell-surface markers by methods known to those skilled in the art. In some embodiments, an agent that specifically bind to a cell-surface marker, such as an antibody or antigen-binding fragment, is labelled with a tag to facilitate the isolation and detection of population of cells of the interest. As used herein, the terms "label" or "tag" refer to a composition capable of producing a detectable signal indicative of the presence of a target, such as, the presence of a specific cell-surface marker in a sample. Suitable labels includefluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods to isolate and detect the cells. Non-limiting examples of fluorescent labels or tags for labeling the agents such as antibodies for use in the methods of invention include Hydroxy coumarin, Succinimidyl ester, Aminocoumarin, Succinimidyl ester, Methoxycoumarin, Succinimidyl ester, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R-Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, PerCPeFluor 710, PE-CF594, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, BV 785, BV711, BV421, BV605, BV510 or BV650.

[0032] As used herein, the term “predetermined reference value” refers to a threshold value or a cutoff value. A "threshold value", “reference value” or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and / or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the level of the markers of the invention in properly banked historical patient samples may be used in establishing the predetermined corresponding reference value. In some embodiments, the predetermined corresponding reference value is the median measured in the population of the patients for the marker of in the invention. In some embodiments, the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of the marker of the invention in a group of reference, one can use algorithmic analysis for the statistic treatment of the levels determined in samplesto be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc. In some embodiments, the frequency of the Tfr cells, in particular CD3+CD4+CD45RA CXCR5+Foxp3+cells, is 1, 2, 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% higher as compared to the predetermined reference value. In some embodiments, the predetermined reference value is the frequency of the Tfr cells, in particular CD3+CD4+CD45RA CXCR5+Foxp3+cells, in a population of cells in a first sample obtained from the subject. In some embodiments, the first sample is obtained from the subject at least 1, 2, 3, 4 or 5 weeks before the second one. In some embodiments, the first sample is obtained from the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months before the second one. In some embodiments, the first sample is obtained from the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years before the first one. In some embodiments, the predetermined reference value is the frequency of the Tfr cells, in particular CD3+CD4+CD45RACXCR5+Foxp3+cells, in a population of cells in a sample obtained when the subject was relapsing. In some embodiments, the frequency of the Tfr cells, in particular CD3+CD4+CD45RA CXCR5+Foxp3+cells, is 1, 2, 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98,99 or 100% lower as compared to the frequency of Tfr cells, in particular CD3+CD4+CD45RA' CXCR5+Foxp3+cells, in a population of cells in a sample obtained when the subject was relapsing. In some embodiments, the predetermined reference value is the frequency of the Tfr cells, in particular CD3+CD4+CD45RACXCR5+Foxp3+cells, in a population of cells in a sample obtained when the subject was remitting. In some embodiments, the frequency of the Tfr cells, in particular CD3+CD4+CD45RA CXCR5+Foxp3+cells, is 1, 2, 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% higher as compared to the frequency of Tfr cells, in particular CD3+CD4+CD45RACXCR5+Foxp3+cells, in a population of cells in a sample obtained when the subject was remitting.

[0033] In some embodiments, the present invention relates to a kit for predicting or diagnosing active disease in a subject suffering from multiple sclerosis, said kit comprising reagents for detecting the frequency of Tfr cells in a population of cells in a sample obtained from said subject. In some embodiments, the present invention also relates to a kit for predicting or diagnosing active disease in a subject suffering from multiple sclerosis, said kit comprising reagents for detecting the frequency of CD3+CD4+CD45RA CXCR5+Foxp3+cells in a population of cells in a sample obtained from said subject.

[0034] As used herein, the term “kit” refers to a collection of components, materials or tools that are packaged together and designed to perform a specific function or achieve a specific purpose as claimed in the invention. As used herein, the term “reagent” refers to a chemical substance or compounds that is specifically used in a chemical reaction to detect or measure the biomarkers of the present invention (e.g. an antibody, an immunoglobulin, an aptamer).

[0035] Methods of treating

[0036] In another aspect, the present invention relates to a method of treating a subject suffering from multiple sclerosis, said method comprising the step of predicting or diagnosing active multiple sclerosis in said subject by determining the frequency of Tfr cells in a population of cells in a sample obtained from the subject. In some embodiments, the present invention relates to a method of treating a subject suffering from multiple sclerosis, said method comprising the step of predicting or diagnosing active multiple sclerosis in said subject by determining the frequency of CD3+CD4+CD45RA CXCR5+Foxp3+cells in a population of cells in a sample obtained from the subject.In some embodiments, the present invention relates to a method of treating a subject suffering from multiple sclerosis, said method comprising the steps of:

[0037] - Determining the frequency of Tfr cells in a population of cells in a sample obtained from the subject;

[0038] Concluding that the subject suffers from active multiple sclerosis when the frequency of Tfr cells is higher than a predetermined reference value; and

[0039] Administering said subject with a therapeutically effective amount of a treatment against MS.

[0040] In some embodiments, the present invention relates to a method of treating a subject suffering from multiple sclerosis, said method comprising the steps of:

[0041] Determining the frequency of CD3+CD4+CD45RA CXCR5+Foxp3+cells in a population of cells in a sample obtained from the subject;

[0042] Concluding that the subject suffers from active multiple sclerosis when the frequency of CD3+CD4+CD45RACXCR5+Foxp3+cells is higher than a predetermined reference value; and

[0043] Administering said subject with a therapeutically effective amount of a treatment against MS.

[0044] In some embodiments, the present invention relates to a method of monitoring a treatment of a subject suffering from multiple sclerosis, said method comprising the steps of:

[0045] Determining the frequency of Tfr cells, in particular CD3+CD4+CD45RA' CXCR5+Foxp3+cells, in a population of cells in a first sample obtained from the subject; Administering said subject with a therapeutically effective amount of a treatment against multiple sclerosis;

[0046] Determining the frequency of Tfr cells, in particular CD3+CD4+CD45RA' CXCR5+Foxp3+cells, in a population of cells in a second sample obtained from the subject;

[0047] Concluding that the treatment is efficient when the frequency of Tfr cells in the second sample obtained from said subject is lower than the frequency of Tfr cells in the first sample obtained from said subject.

[0048] In some embodiments, the first sample is obtained from the subject at least 1, 2, 3, 4 or 5 weeks before the second one. In some embodiments, the first sample is obtained from the subject atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months before the second one. In some embodiments, the first sample is obtained from the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years before the second one.

[0049] In some embodiments, the second sample is obtained from the subject at least 1, 2, 3, 4 or 5 weeks after the administration of the treatment against said multiple sclerosis. In some embodiments, the second sample is obtained from the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after the treatment against said multiple sclerosis. In some embodiments, the second sample is obtained from the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years after the treatment against said multiple sclerosis.

[0050] As used herein, the terms “treating”, “treatment” or “therapy” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disorder or suspected to have contracted the disorder as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The term encompasses both drug administration and non-drug treatment. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g.,administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0051] As used herein, the term “monitoring” refers to the process of assessing, tracking and analysing one or more parameters associated with a treatment over a specified period, in order to determine its effectiveness and safety. Typically, the treatment can either be continued if the frequency of Tfir indicates that the treatment is achieving the desired therapeutic effect (e.g. frequency of Tfr cells lowering) or be discontinued if the frequency of Tfr indicates a lack of efficacy (e.g. frequency of Tfr cells increasing).

[0052] As used herein, the term "administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0053] In some embodiments, the treatment against multiple sclerosis is selected from the list comprising : disease modifying therapies such as interferons, glatiramer acetate, fmgolimod, teriflunomide, dimethyl fumarate, ozanimod, natalizumab, ocrelizumab, alemtuzumab, ofatumumab; corticosteroids (e.g. methylprednisolone, prednisone), in particular for relapse management; baclofen, tizanidine, amantadine, modafinil, gabapentin, pregabalin, oxybutynin, desmopressin, antidepressants, in particular for symptoms management; and / or rehabilitation and supportive therapies such as physical therapy, occupational therapy, speech therapy or cognitive therapy. The term also includes hematopoietic stem cell transplantation; remyelination and neuroprotection therapies such as anti -LINGO- 1 antibodies (e.g. opicinumab), ibudilast and biotin; BTK inhibitors (e.g. tolebrutinib, evobrutinib); etrasimod; siponimod; mocravimod; cenerimod; icanbelimod; amiselimod: tamuzimob; vibozilimod; BMS-542; ethoximod; BMS-520; udifitimod; GSK1842799; TE-5126; GSK-2263167; and / or ponesimod. In some embodiments, the treatment against multiple sclerosis is a S1PR2 agonistsuch as CYM-5520. In some embodiments, the treatment against multiple sclerosis is a VLA4-antagonist such as natalizumab.

[0054] In some embodiments, the present invention also relates to a method of treating a subject suffering from multiple sclerosis, said method comprising a step of administering said subject with a S1PR2 agonist.

[0055] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0056] FIGURES:

[0057] Figure 1. Tfr frequency and Tfr / Tfh ratio are higher in patients with active MS. PBMCs from HD and RRMS patients were stained and analyzed by flow cytometry in order to characterized the proportion of Tfh (CD3+CD4+CD45RACXCR5+Foxp3 ) and Tfr (CD3+CD4+CD45RACXCR5+Foxp3+) among CD4+T-cells (A). RRMS were stratified as ‘relapse’ if they had a relapse in the last 3 months before sampling, and as ‘remission’ if the last relapse was over 3 months before sampling (B). Longitudinal analysis of PBMC of RRMS patients before (active) and at 1 year of Natalizumab (inactive). Patients were characterized as inactive RRMS since they all responded to the treatment based on NEDA-3 criteria. PBMCs were analyzed by flow cytometry in order to characterize the proportion of Tfh (CD3+CD4+CD45RACXCR5+Foxp3 ) and Tfr (CD3+CD4+CD45RA CXCR5+Foxp3+) (C).

[0058] Tfr promote the severity of rhMOG-EAE. WT, CD4creBcl6fl / flor Tfr-deficient mice were immunized lOOpg of rhMOG emulsified in CFA and injected with pertussis toxin (PTX) at day 0 and 2 in order to induce EAE. The clinical score was assessed daily up to day 23 after immunization. Values are the mean of 33 (WT), 9 (CD4creBcl6fl / fl) and 35 (Tfr-deficient) mice, bars represent the SEM. Cumulative scores are presented on the right panels. Statistical analyses were performed using the repeated measure ANOVA test for the clinical score or Mann-Whitney test for cumulative score, ns = not significant, *p < 0.05, **p < 0.01.

[0059] Tfr increase the production of proinflammatory cytokines by CD4+T-cells in the brain at the peak of EAE. WT or Tfr-deficient mice were immunized with rhMOG at day 0 and injected with PTX at day 0 and day 2. Brains were analyzed at day 14 by flow cytometry.Total CD4+T-cells (A) are characterized as CD45+CD3+CD4+cells, rhMOG-specific T-cells (B) as CD45+CD3+CD4+CD44+I-Ab / rhMOG tetramer . The production of IFNy and IL17-Aby conventional T-cells (CD45+CD3+CD4+CD44+Foxp3‘) from the brain (C) was assessed by flow cytometry after 4 hours of PMA-ionomycin restimulation. Cells producing only fFNy are IFNy4, the ones producing only IL17-A are IL17-A4and cells producing both cytokines are double producers (DP). Each dot represents an individual mouse (n=9 for WT and n=9 for Tfr-deficient) from two independent experiments. Statistical analyses were performed using the Mann-Whitney test, ns = not significant, *p < 0.05, **p < 0.01.

[0060] Tfr promote the migration to the brain of B-cells with an APC function at the peak of EAE. WT or Tfr-deficient mice were immunized with rhMOG at day 0 and injected with PTX at day 0 and day 2. Brains were analyzed at day 14 by flow cytometry (A-B). B-cells (A) are gated on CD45+CD19+, activated B-cells on CD45+CD19+IgD‘, T-cells on CD45+CD3+and DC on CD45+CD1 lc+I-Abhl(B). Each dot represents an individual mouse (n=9 for WT and n=9 for Tfr-deficient) from two independent experiments. Statistical analyses were performed using the Mann-Whitney test, ns = not significant, **p < 0.01. (C) Spleens and brains were processed to sort DC (CD45+CDllc+I-Abhl) and activated B-cells (CD45+CD19+IgD ), which were then cocultured with activated TCR-transgenic (2D2) MOG-specific T-cells (CD3+CD4+CD44+) at a 1 : 1 ratio. T-cell reactivation was assessed by an ELISpot against fFNy. Each dot represents one well, from two independent experiments. Statistical analyses were performed using the Mann-Whitney test, ns = not significant, *p < 0.05, ***p < 0.001, ****p < 0.0001.

[0061] Tfr decrease the number of GC-B cells in draining lymph nodes by inducing the downregulation of S1PR2. WT or Tfr-deficient mice were immunized with rhMOG at day 0 and injected with PTX at day 0 and day 2. dLN were analyzed at day 7 by flow cytometry. Activated B-cells (A-B) are gated on CD45+B220+CD19+, and GC-B cells (C) are gated on CD45+B220+CD19+IgD CD138 Bcl6 Fas Each dot represents an individual mouse (n=8 for WT and n=7 for Tfr-deficient) from two independent experiments. Statistical analyses were performed using the Mann-Whitney test, ns = not significant, **p < 0.01, ***p <0.001, ****p < 0.0001.

[0062] Inhibiting S1PR2 signaling promotes EAE severity in absence of Tfr. WT or Tfr-deficient mice were immunized with rhMOG and injected with PTX at day 0 and 2 in order toinduce EAE. Mice were treated daily for a week with JTE-013 or vehicle between day 5 and day 12 post-immunization. The clinical score was assessed up to day 14 after immunization (A), each point represents the mean value of 5 mice (WT + JTE-013), 10 mice (WT + vehicle and Tfr-deficient + vehicle) or 15 mice (Tfr-deficient+ JTE-013). Bars represent the SEM. The graph was analyzed by repeated measure ANOVA. ****p < 0.0001. Cumulative scores are the sum of all scoring by mouse. Brains were analyzed at day 14 by flow cytometry. Total B-cells (B) are gated on CD45+CD19+cells. The production of IFNy et IL17-Aby conventional T-cells (CD45+CD3+CD4+CD44+Foxp3 ) (C) is assessed by flow cytometry after 4 hours of PMA-ionomycin restimulation. Cells producing only IFNy are IFNy+, the cells producing only IL17-A are IL17-A+and cells producing both cytokines are DP. All graphs were analyzed by Mann-Whitney test, ns = not significant, *p < 0.05, ****p < 0.0001.

[0063] EXAMPLE:

[0064] Material and Methods

[0065] Human samples. Samples from RRMS patients and healthy donors (HD) were obtained from MS expert center of Rennes (France). MS patients were diagnosed according to the McDonald 2017 criteria. Patients had no disease modifying drug for a period of at least 1.5 years before sampling. This study was approved by Rennes institutional ethical committee (ethical committee n° 19.131 -bis). A second set ofPBMC derived from MS patients has been analyzed. These PBMC were isolated from Natalizumab responding RRMS patients before and after one year of treatment. The response was defined after 2 years of Natalizumab using the NEDA-3 (no evidence of disease activity 3) criteria. The NEDA-3 includes three parameters: no relapse, no new lesion on T2-weighted sequence nor gadolinium-enhancing lesion during MRI monitoring, and no disability progression assessed by the EDSS score. In accordance with law n° 2004-806 of August 9, 2004 relating to public health policy, this research (NCT00942214) was approved by the Comite de Protection des Personnes (CPP) Sud Ouest et Outre Mer II, authorized on 09 / 02 / 2009, and was the subject of a declaration to the Commission Nationale Informatique et Libertes (CNIL). All donors provided written informed consent in compliance with the Declaration of Helsinki.

[0066] Mice. CD4cre, Foxp3cre, Bcl6fl / flmice were purchased from the Jackson laboratory. 2D2 mice were a gift from Dr Anne Dejean. All mice were bred in UMS006 and only females of 8-12 weeks of age were used for experimental procedures. Mouse experimental protocols wereapproved by the French ‘Ministere de 1’Enseignement Superieur et de la Recherche’ (ethical review N° MP / 19 / 58 / 06 / 12).

[0067] Induction of EAE. Complete Freund’s adjuvant (CFA), Mycobacterium butyricum (Mtb) and Pertussis toxin (PTX) from Bordetella pertussis were from Sigma-Aldrich, rhMOG from Eurogentec. Mice were immunized subcutaneously on day 0 at the base of tail with 200pL containing lOOpg of rhMOG emulsified in CFA containing 2mg / mL Mtb. PTX injections were done intravenously with 2 pg in lOO L PBS at day 0 and 4 g at day 2. Disease was monitored daily by the following criteria: 0, no symptom; 1, tail paralysis; 2, hind limb paresis; 3, complete hind limb paralysis; 4, forelimbs paralysis; and 5, moribund.

[0068] In vivo treatment. Mice received every day one intraperitoneal injection of 8mg / kg of JTE-013 (Sigma) in PBS 12.5% DMSO from day 5 to 12 after immunization. Control mice received injections of vehicle (PBS 12.5% DMSO).

[0069] ELISPOT. Anti-IFNy precoated plates (Mabtech) were washed 4 times with sterile PBS, RPMI with 10% FBS was added for 30min at room temperature. Plates were washed, and cells were added with or without 4pg of MOG35-55 and put in culture at 37°C 5% CO2 for 42 hours. Cells were then removed and we added 1 pg / mL of anti-IFN > (R4-6A2, biotinyled, Mabtech) for 2 hours at RT. After one wash, plates were incubated for 1 hour at RT with ALP-streptavidin (Mabtech). After one wash, filtrated BCIP / NBT was added for 15 minutes at RT in the dark. The reaction was stopped by putting tap water. Plate reading was performed with Immunospot Analyzer (C.T.L).

[0070] ELISA. rhMOG-specific IgG (Southern Biotechnology Associates) and total IgA were detected in sera by ELISA. ELISA plates (Thermo scientific) were coated overnight at 4°C with 10 pg / mL rhMOG (Eurogentec) or 2.5pg / mL anti-IgA (polyclonal rat, BD Biosciences). After being washed, plates were saturated for lh30 with PBS containing 0.05% Tween (Sigma-Aldrich) and 1% BSA (Dutscher). Then plates were washed and incubated for 2 hours with serum dilution. After 2 hours, plates were washed again and incubated for 2 hours with either anti-IgG-biotin (SouthernBiotech) or anti IgA-biotin (SouthemB iotech). Plates were washed and incubated for 15 minutes with HRP-streptavidin (SouthernBiotech). Finally, plates were washed, and we added sigma Fast OPD (Sigma) until color change. Optical density (OD) was read on a VersaMax Microplate Reader (Molecular devices) with X=490 nm.Flow cytometry analysis and cell sorting. dLN and brain were collected from mice sedated with lethal dose of ketamine / xylasine followed by a cardiac perfusion of lOmL of PBS. Brains were digested with collagenase / DNase I (Roche) for 30 min at 37°C. After digestion, brain cells were resuspended in 30% Percoll (GE healthcare) to enrich in hematopoietic cells. Other cell suspensions were prepared in PBS / 2% FCS, 5mM EDTA. The following mAbs were used: anti-CDllb (MI / 70, BUV395, BD Biosciences) anti-CD4 (RM4-5, BUV496, BD Biosciences), anti-CDllc (HL3, BUV615, BD Biosciences), anti-B220 (RA3-6B2, BUV615, BD Biosciences), anti-IgD (1 l-26c.2a, BV421, Biolegend), anti-CD45 (30-F11, Bv510, Biolegend) anti-CD44 (IM7, BV570, Biolegend), anti-CD138 (281-2, BV605, Biolegend), anti-CD3 (500A2, BV785, Biolegend), anti-Bcl6 (KI 12-91, Alexa Fluor 488, BD Biosciences), anti-IgD (ll-26c, Alexa Fluor 488, BD Biosciences) anti-PD-1 (J43, PerCP Cy5.5, Invitrogen), anti-I-Ab(AF6-120.1, PerCP Cy5.5, Invitrogen) anti-Bcl6 (KI 12-91, PECF594, BD Biosciences), anti-CD19 (1D3, PECF594, BD Biosciences), anti-Fas (Jo2, PeCy7, BD Biosciences), anti-CXCR5 (REA215, APC, Miltenyi), anti-SlPR2 (F-3, APC efluor 660, Santa Cruz Biotechnology) and anti-B220 (RA3-6B2, APC Cy7, BD Biosciences). Peptide rhMOG (MOG38-49 GWYRPPFSRVVH)-I-Abtetramers were obtained from NIH Tetramer core facility. For intracellular staining, cell suspensions were fixed and permeabilized using BD Fixation / Permeabilization kit. For cytokine production, cells were restimulated for 4 hours with 50ng / mL of PMA (Sigma), l,5pg / mL of ionomycine (Sigma), lOpg / mL of brefeldin A (eBioscience) and 2pM of monensin (eBioscience) in complete RPMI. Cells were then washed and stained with anti-CD4 (RM4-5, BUV496, BD Biosciences), anti-CD45 (30-F11, BV510, Biolegend), anti-CD44 (IM7, BV570, Biolegend), anti-IL17-A (TC11-18H10, BV711, Biolegend), anti-CD3 (500A2, BV785, Biolegend), anti-IFND (XMG1.2, PECF 594BD Biosciences), anti-Foxp3 (FJK-16s, PeCy7, Invitrogen). Before permeabilization cells were stained with Fixable Viability stain 440UV (BD Biosciences). Human cells were stained with anti-CD3 VioBlue (BW264 / 56, Miltenyi Biotec), anti-CD3 BUV395 (UCHT1, BD Biosciences), anti-CD45RA PE-Vio615 (REA562, Miltenyi Biotec), anti-CD45RA BV605 (HI100, Biolegend), anti-CXCR5 PeVio770 (REA103, Miltenyi Biotec), anti-CXCR5 APC (REA103, Miltenyi Biotec), anti-FoxP3 APC (REA1253, Miltenyi Biotec), anti-Foxp3 PE (236A / E7, BD Biosciences), anti-CD4 BV786 (SK3, BD Biosciences), anti CD4 BUV496 (SK3, BD Biosciences), and anti-CD8 BUV805 (SKI, BD Biosciences). Cells were incubated at RT for 30 min. Before permeabilization cells were stained with Fixable Viability Dye eF506 (Invitrogen). Stained cells were acquired using a Fortessa flow-cytometer or a BD FACSymphony™ A5 Cell Analyzer (BD Biosciences, San Jose, CA), sorted usingFACSARIA-SORP (BD Biosciences) or FACSARIA-FUSION (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR). Doublets and dead cells were excluded using appropriate FSC / SSC gates and the viability marker.

[0071] Statistical analysis. Differences between variables were evaluated using the non-parametric Mann-Whitney test or Wilcoxon test. All statistical analyses were carried out with GraphPad Prism 10.0 software (San Diego, California, USA). / ?-values less than 0.05 were considered statistically different.

[0072] Results

[0073] Frequency of circulating Tfr in patients with MS follows clinical activity. In order to evaluate the contribution of Tfr to MS pathogenesis, we took advantage of two cohorts of patients with MS. The first one was composed of patients with RRMS, among whom 44 were drug naive and 4 not exposed to immunomodulatory or immunosuppressive drugs for at least 1.5 years (RRMS n=48; data not shown). We studied the peripheral blood mononuclear cells (PBMC) of these patients in comparison to those from age- and sex-matched HD (n=31). We studied the frequencies of Tfr and Tfh among total CD4+T-cells (data not shown). We found that the Tfr / Tfh ratio was similar between patients as compared to HD (FigurelA left, 0.058±0.008 in HD; 0.071±0.012 in RRMS patients). This correlated with similar frequencies of circulating Tfh and Tfr between patients as compared to HD (Figure 1A middle, 9.35%±0.55 in HD; 9.83%±0.42 in RRMS patients; FigurelA right, 0.53%±0.07 in HD; 0.88%±0.22 in RRMS patients). Thus, the Tfr / Tfh ratio could not discriminate HD from RRMS patients. Interestingly, when patients were stratified based on their MS clinical activity, i.e. patients in relapse (n=26) vs. patients in remission (n=22), we found that the Tfr / Tfh ratio was decreased in RRMS patients in remission (FigurelB left, 0.0943±0.02 in RRMS patients in relapse; 0.0441±0.009 in RRMS patients in remission), a phenomenon that relied on the decrease of the frequency of circulating Tfr (Figure IB right, 0.98%±0.25 in RRMS patients in relapse; 0.39%±0.07 in RRMS patients in remission) and not a modification of the Tfh frequency (Figure IB middle, 9.70%±0.52 in RRMS patients in relapse; 9.99%±0.70 in RRMS patients in remission). The second group we investigated was a prospective cohort of RRMS patients followed longitudinally during the course of an efficient treatment with Natalizumab (n=20, data not shown). Thus, we studied the frequencies of Tfr and Tfh among total CD4+T-cells (data not shown) at treatment initiation, when MS was clinically active, and one year after treatment, when MS was inactive both clinically and radiologically. We found that theTfr / Tfh ratio decreased, similarly to clinical activity in these Natalizumab treated MS patients (Fig.lC left, 0.06±0.012 in active MS, 0.02±0.003 in inactive MS). Again, this correlated with the decrease of the frequency of circulating Tfr (Figure 1C right, 1 ,27%±0.19 in active MS vs.

[0074] 0.42%±0.10 in inactive MS) and not a modification of the Tfh frequency (Figure 1C middle, 22.70%±0.93 in active MS vs. 18.35%±1.88 in inactive MS). Thus, in two different cohorts of MS patients, frequency of circulating Tfr in MS patients reflected the ongoing clinical activity, suggesting that Tfr may contribute to the clinical activity of MS.

[0075] Tfr promote the severity of autoimmune encephalomyelitis in mice. To further evaluate the functional role of Tfr during autoimmune neuroinflammation and their impact on encephalitogenic B- and T-cells, we analyzed the induction and severity of EAE in presence or absence of Tfr. To this end, we crossed the mouse strain in which the ere recombinase is knocked into the Foxp3 gene locus with the mouse strain with the conditional Bcl6 allele38. Only Foxp3cre / creBcl6fl / flfemale mice were used as it has been demonstrated that mice with this genotype lacked Tfr while Foxp3cre / +Bcl6fl / flfemale mice had comparable Tfr than WT mice29. We induced EAE in Foxp3+ / +Bcl6fl / fl(referred to as WT) and Tfr-deficient mice by immunization with the rhMOG. Indeed, it has been shown that rhMOG immunization can only induce EAE in presence of B-cells37, thus a mouse model that recapitulates features observed in MS relapses. Notably, mice lacking AID expression are resistant to rhMOG-induced EAE, suggesting that GC maturation processes are critical processes in the pathogenesis of EAE39. Here, we observed that CD4creBcl6fl / flmice, which lack both Tfh and Tfr, were resistant to rhMOG-induced EAE, thereby demonstrating the significance of GC reactions in the development of this disease (Figure 2). Moreover, we found that selective reduction of Tfr (data not shown) led to a less severe EAE with significant decrease for the clinical score at the peak of the disease from days 12-18 post-immunization (Figure 2). This decrease in EAE severity resulted in a notably reduced cumulative score in Tfr-deficient mice (Figure 2).

[0076] Interestingly, this difference could not be accounted for by a difference in serum anti-rhMOG IgG at the peak of the disease day 14 post-immunization nor by a difference in total serum IgA, which have been shown to have a protective role in the context of EAE40(data not shown).

[0077] Thus, in accordance with the observation in our MS cohorts, we demonstrated that Tfr cells in mice promote clinical severity in autoimmune encephalomyelitis.

[0078] Tfr promote the production of pro-inflammatory cytokines by brain-infiltrating T-cells during EAE in mice. Since EAE is mediated in part by pathogenic T-cells that infiltrate theCNS, we assessed whether the lowered EAE severity in Tfr-deficient mice was due to a reduced infiltration of these cells. We thus monitored by flow cytometry brain CD4+T-cells at 14 days post-immunization, the peak of the disease, from rhMOG-induced EAE WT and Tfr-deficient mice. Frequencies of CD4+T-cells were similar in the two groups of mice (Figure 3A). Further, using MHC class IVpeptide tetramers, we focused on rhMOG-specific CD4+T-cells and observed no differences between WT and Tfr-deficient mice for their frequency among CD4+T-cells (Figure 3B). In contrast, re-stimulated brain conventional CD4+T-cells had a reduced production of IFNy and IL17-A in Tfr-deficient mice as compared to WT mice (Figure 3C).

[0079] Notably, when we studied CD4+T-cells in the dLN 7 days post-immunization, we observed no significant differences between WT and Tfr-deficient mice regarding frequency of CD4+T-cells (data not shown), rhMOG-specific CD4+T-cell frequency (data not shown) and cytokine production by conventional CD4+T-cells (data not shown). Overall, despite the similarity in the extent and cytokine production of the CD4+T-cell response in the dLN after EAE induction in WT and Tfr-deficient mice, the brain-infiltrating CD4+T-cells produce less pro-inflammatory cytokines in Tfr-deficient mice, which correlates with lower EAE severity.

[0080] Brain-infiltrating B-cells reactivate in situ T-cells during EAE in mice. In the course of EAE, encephalitogenic CD4+T-cells become pathogenic after in situ reactivation41. We thus assessed whether the decreased production of pro-inflammatory cytokines by brain CD4+T-cells in Tfr-deficient mice could be due a defect in antigen presentation. First, we evaluated the frequencies of microglia and conventional dendritic cells (DC) in the brain of EAE induced WT and Tfr-deficient mice at the peak of the disease as well as the MHC class II molecule I-Aband the co-stimulatory molecule CD86 expression levels at their surface. No significant differences were observed between the two groups (data not shown). B-cells were shown to contribute to EAE pathology by reactivating encephalitogenic T-cells through antigen presentation37, we thus turned our attention to these cells. We found a dramatic decrease in the frequency of brain B-cells in Tfr-deficient mice at the peak of EAE as compared to WT mice (Figure 4A). Braininfiltrating activated B-cells at the peak of the disease in WT mice expressed high levels of I-Aband CD86 closely similarly to DC (Figure 4B). Using an ELISPOT approach, as performed by others with brain DC from MOG35-55-EAE induced WT mice42, we found that MOG35-55 peptide-loaded DC and B-cells from the brain of rhMOG-EAE induced WT mice at the peak of the disease had a similar capacity to reactivate and induce IFN > production by pre-activated MOG35 -55-specific 2D2 T-cells (Figure 4C). More importantly, without loading of the cells with MOG35-55 peptide, we observed that brain B-cells from rhMOG-EAE induced WT mice atthe peak of the disease had the capacity to induce IFNy production by 2D2 T-cells while splenic B-cells from the same mice did not (Figure 4C). Thus, in presence of Tfr, B-cells migrate to the brain of rhMOG-EAE induced mice where they have the capacity to capture, process and present antigen through MHC class II molecules to T-cells and, thereby, induce their cytokine production.

[0081] GC-B cells are trapped in dLN of EAE induced Tfr-deficient mice. To better understand why B-cells do not migrate to the brain of rhMOG-EAE induced Tfr-deficient mice, we studied the B-cell response at day 7 post-immunization in the dLN from WT and Tfr-deficient mice after EAE induction. We found that the frequencies of total B-cells were similar in EAE-induced WT and Tfr-deficient mice (data not shown). In contrast, the frequency of activated B-cells was greater in Tfr-deficient mice (Figure 5A). This increase in activated B-cells in Tfr-deficient mice was associated with an increase in the frequency of Tfh (data not shown), as already observed in another autoimmune context in Tfr-deficient mice30. Moreover, while CXCR5 expression at the surface of activated B-cells in WT and Tfr-deficient mice was similar, the expression level of Bcl6 was significantly higher in activated B-cells from Tfr-deficient mice (Figure 5B), suggesting a stronger GC phenotype for activated B-cells in absence of Tfr cells. Interestingly, activated B-cells in Tfr-deficient mice expressed higher levels of S1PR2, the receptor that allows B-cell confinement to GC20,21(Figure 5B). This increased GC phenotype among activated B-cells in Tfr-deficient mice translated in an increase in GC-B cells (Figure 5C). In conclusion, more GC-B cells accumulate in the dLN of Tfr-deficient mice after EAE induction, a phenomenon associated with the higher cell surface expression of S1PR2.

[0082] S1PR2 antagonist restores EAE severity in Tfr-deficient mice by promoting B-cell migration to the brain. To assess mechanistically whether higher S1PR2 expression at the surface of GC-B cells accounts for the decreased EAE severity in Tfr-deficient mice, we took advantage of the S 1PR2 antagonist, JTE-013. We daily treated mice undergoing EAE with JTE-013 or vehicle alone from days 5-12 after immunization. Vehicle-treated Tfr-deficient mice had a lower EAE severity than vehicle-treated WT mice as observed before. Moreover, JTE-013-treated WT mice had a similar EAE severity and cumulative score than vehicle-treated WT mice (Figure 6A). Strikingly, JTE-013 -treated Tfr-deficient mice developed an EAE with a similar course and cumulative score as JTE-treated WT mice (Figure 6A). Thus, inhibiting S1PR2 signaling allows the Tfr-deficient mice to develop an EAE as severe as that of WT mice. This increase in EAE-severity was not due to an increase of total brain CD4+T-cells norrhMOG-specific CD4+T-cells in JTE-013 -treated Tfr-deficient mice (data not shown).

[0083] However, brain B-cells at the peak of the disease were increased in Tfr-deficient mice after JTE-013 treatment to the level observed in vehicle-treated WT mice (Figure 6B). This increase in B-cell migration to the brain had an impact on cytokine production by T-cells since the frequency of conventional CD4+T-cells producing IFNy was enhanced in JTE-013 -treated Tfr-deficient mice as compared to vehicle-treated Tfr-deficient mice and reached a similar level than in vehicle-treated WT mice (Figure 6C). Altogether, we demonstrated that, during EAE, higher S1PR2 expression at the surface of activated B-cells in Tfr-deficient mice favors their retention in GC in the dLN, thereby preventing their migration to the brain and subsequent in situ restimulation of encephalitogenic T-cells and, ultimately, blunting EAE severity.

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Claims

- 26 -CLAIMS:

1. A method for predicting active disease in a subject suffering from multiple sclerosis, said method comprising the step of determining the frequency of Follicular Regulatory T (Tfir) cells in a population of cells in a sample obtained from the subject.

2. A method for diagnosing active disease in a subject suffering from multiple sclerosis, said method comprising the steps of:- Determining the frequency of Tfir cells in a population of cells in a sample obtained from the subject; andConcluding that the subj ect suffers from an active multiple sclerosis when the frequency of Tfr cells is higher than a predetermined reference value.

3. A kit for predicting or diagnosing active disease in a subject suffering from multiple sclerosis, said kit comprising reagents for detecting the frequency of Tfr cells in a population of cells in a sample obtained from said subject.

4. A method of treating a subject suffering from multiple sclerosis, said method comprising the steps of:- Determining the frequency of Tfr cells in a population of cells in a sample obtained from the subject;Concluding that the subject suffers from active multiple sclerosis when the frequency of Tfr cells is higher than a predetermined reference value; and Administering said subject with a therapeutically effective amount of a treatment against said multiple sclerosis.

5. A method of monitoring a treatment of a subject suffering from multiple sclerosis, said method comprising the steps of:- Determining the frequency of Tfr cells in a population of cells in a first sample obtained from the subject;Administering said subject with a therapeutically effective amount of a treatment against said multiple sclerosis;- Determining the frequency of Tfr cells in a population of cells in a second sample obtained from the subject;Concluding that the treatment is efficient when the frequency of Tfir cells in the second sample obtained from said subject is lower than the frequency of Tfir cells in the first sample obtained from said subject.

6. The method according to claim 4 or 5, wherein the treatment against said multiple sclerosis is a VLA4-antagonist.

7. The method according to claim 6, wherein the VLA4-antagonist is natalizumab.

8. The method or kit according to any of claims 1 to 7, wherein the sample obtained from the subject is a blood sample.

9. The method or kit according to any of claims 1 to 8, wherein the sample obtained from the subject is a sample of peripheral blood mononuclear cells (PBMC).

10. The method or kit according to any of claims 1 to 9, wherein the Tfr cells are CD3+CD4+CD45RA CXCR5+Foxp3+cells.

11. The method or kit according to any of claims 1 to 10, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.

12. The method or kit according to any of claims 1 to 11, wherein the subject is asymptomatic.

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