WO2020043693A1 - Diagnosis of multiple sclerosis - Google Patents

Abstract

The present invention relates to a method of diagnosing a subject with multiple sclerosis comprising determining different cell ratios in a test cerebrospinal fluid (CSF) sample obtained from said subject, wherein the combination of said specific cell ratios is indicative for whether or not said subject suffers from multiple sclerosis. Further, the present invention relates to a data processing system comprising a processor configured to perform the steps of said method above, a flow cytometry device capable of detecting the specific cell levels in a test CSF sample comprising the defined data processing system, a computer program comprising instructions to cause the data processing system or the flow cytometry device to execute the steps of the defined method, as well as a computer-readable medium having stored thereon the computer program as defined. The present invention also relates to a kit and an agent for use in the treatment of multiple sclerosis.

Description

Diagnosis of multiple sclerosis

The present application claims the benefit of priority of LU Patent Application LU 100915 filed 29 August 2018, the content of which is hereby incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD OF THE INVENTION

[001] The present invention relates to a method of diagnosing a subject with multiple sclerosis comprising determining different cell ratios in a test cerebrospinal fluid (CSF) sample obtained from said subject, wherein the combination of said specific cell ratios is indicative for whether or not said subject suffers from multiple sclerosis. Further, the present invention relates to a data processing system comprising a processor configured to perform the steps of said method above, a flow cytometry device capable of detecting the specific cell levels in a test CSF sample comprising the defined data processing system, a computer program comprising instructions to cause the data processing system or the flow cytometry device to execute the steps of the defined method, as well as a computer-readable medium having stored thereon the computer program as defined. The present invention also relates to a kit and an agent for use in the treatment of multiple sclerosis.

BACKGROUND OF THE INVENTION

[002] Multiple sclerosis (MS) is a chronic inflammatory, demyelinating disorder of the CNS that can cause substantial permanent disability. The diagnosis of multiple sclerosis (MS) is based on clinical, radiological, and laboratory findings. MS is commonly considered as an autoimmune disease of the CNS based on histopathological evidence of inflammatory infiltrates in MS lesions, positive effects of immunomodulatory therapies and results from genome wide association studies.

[003] Evidence supports an involvement of both T cells and B cells in MS, but the relative contribution of each cell type remains unknown. On the one hand, an expansion of B cells and production of immunoglobulins occurs locally in the CNS and B cell depleting therapies are effective in MS. On the other hand, T cells are the most abundant lymphocytes in MS lesions and T cells transfer an MS-like disease named experimental autoimmune encephalomyelitis (EAE) in experimental animals. It also remains unknown whether T cells modulate B cell function in MS. [004] For the diagnosis of MS cerebrospinal fluid (CSF) is used. CSF is a clear liquid that surrounds and envelops the CNS. It is produced by the choroid plexus in the brain ventricles and circulates into the subarachnoid space until being absorbed into the venous and lymphatic system (Louveau, A. et al. 2015, Nature 523, 337-341 and Engelhardt, B., Vajkoczy, P. & Weller 2017, Nat. Immunol. 18). CSF provides mechanical protection and trophic support and it acts as transport medium for immune cells and antigens. Under healthy conditions, the non-cellular fraction of CSF is mostly an ultra-filtrate of serum with solutes crossing into the CSF in a size- dependent manner. In contrast, CSF cells which derive exclusively from the hematopoietic lineage exhibit a distinct cellular composition compared to blood. The leukocyte concentration in the CSF is 1 ΌOO-fold lower than in the blood and CSF leukocytes show a highly distinct pattern with a predominance of CD4⁺ T lymphocytes and relative lack of myeloid-lineage cells compared to blood. This indicates that the cellular composition of the CSF must be maintained in a controlled manner and is not simply a mirror image of blood. Clinically, CSF provides a unique diagnostic window into immune-related processes in the immediate vicinity of the CNS.

[005] In MS, CSF exhibits several disease-associated changes such as an increased CSF immunoglobulin index and oligoclonal immunoglobulins (i.e. oligoclonal bands) in the CSF (Kivisakk, P. et al., 2003, Proc. Natl. Acad. Sci. U. S. A. 100). In MS tissue lesions, monocytes, microglia and macrophages are most abundant, followed by infiltrated CD8⁺ T cells and CD4⁺ T cells. This discrepancy between tissue and CSF findings remains unresolved, partly because an unbiased characterization of CSF cells is missing.

[006] By now, diagnosing a subject with MS is based on laborious diagnostic procedures such as monitoring MS disease activity by cranial scans, including magnetic resonance imaging (MRI) of the brain.

[007] Therefore, there is a need in the art to provide new, alternative diagnosing methods of multiple sclerosis using CSF, preferably bypassing costly differential diagnosis.

[008] Therefore, the objective of the present invention is to comply with this need.

[009] The solution of the present invention is described in the following, exemplified in the appended examples, illustrated in the figures and reflected in the claims.

SUMMARY OF THE INVENTION

[0010] The present invention deals with generating a comprehensive map of the cellular composition and transcriptional phenotype of CSF cells in MS by determining specific cell ratios in a tested CSF sample obtained from a subject. Evidence of local ongoing B cell maturation correlated with an expansion of T follicular helper (TFH) cells in the CSF - a cell type known to promote B cell responses. Using cutting-edge technology and rare clinical specimen, a new method of diagnosing a subject with multiple sclerosis has been demonstrated using CSF.

[0011] The present invention relates to a method of diagnosing a subject with multiple sclerosis, comprising determining

i) the ratio of the level of NK cells to the level of CD4⁺ T cells;

ii) the ratio of the level of B cells to the level of CD4⁺ T cells;

iii) the ratio of the level of CD8⁺ cells to the level of CD4⁺ T cells, and

iv) the ratio of the level of CD14⁺CD16 monocytes to the level of CD14⁺CD16⁺ monocytes;

in a test cerebrospinal fluid (CSF) sample obtained from said subject, wherein the combination of said ratios i)-iv) is indicative for whether or not said subject suffers from multiple sclerosis.

[0012] Additionally, the present invention may comprise the method as defined elsewhere herein, further comprising determining whether or not an elevated immunoglobulin index and/or oligoclonal bands can be detected in said test CSF sample.

[0013] Further, the present invention may envisage the method as defined elsewhere herein, wherein the level of NK cells, the level of CD4⁺ T cells, the level of B cells, the level of CD8⁺ cells, the level of CD14⁺CD16 monocytes and the level of CD14⁺CD16⁺ monocytes are detected using flow cytometry.

[0014] Also comprised by the present invention may be the method as defined elsewhere herein, wherein detecting the level of NK cells comprises measuring CD45, CD16 and/or CD56; wherein detecting the level of CD4⁺ T cells comprises measuring CD45, CD3 and/or CD4; wherein detecting the level of B cells comprises measuring CD45, CD19 and/or CD138; wherein detecting the level of CD8⁺ T cells comprises measuring CD45, CD3 and/or CD8; wherein detecting the level of CD14⁺CD16 monocytes comprises measuring CD45, CD14 and/or CD16; and/or wherein detecting the level of CD14⁺CD16⁺ monocytes comprises measuring CD45, CD14 and/or CD16.

[0015] Additionally, the present invention may encompass the method as defined elsewhere herein, wherein a binding partner for CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56 and/or CD138 is used, preferably the binding partner is an immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like functions. [0016] Additionally, also comprised may be the method as defined elsewhere herein, wherein said subject is a human.

[0017] Additionally, also comprised may be the method as defined elsewhere herein, wherein said subject is an adult.

[0018] Additionally, also comprised may be the method as defined elsewhere herein, wherein said subject is suspected to suffer from multiple sclerosis (MS).

[0019] The present invention may also comprise the method as defined elsewhere herein further comprising the steps of

a) multiplying

i) the ratio of the level of NK cells to the level of CD4⁺ T cells with an adjustment factor of preferably about 18 to about 74,

ii) the ratio of the level of B cells to the level of CD4⁺ T cells with an adjustment factor of preferably about 25 to about 101 ,

iii) the ratio of the level of CD8⁺ cells to the level of CD4⁺ T cells with an adjustment factor of preferably about 2 to about 8,

iv) the ratio of the level of CD14⁺CD16 monocytes to the level of CD14⁺CD16⁺ monocytes with an adjustment factor of preferably about 0.6 to about 2.3;

b) summing up each adjusted ratio of step a i) - a iv), thereby obtaining a basic composite score; optionally

c) adding a value of 1 to the basic composite score of step b) in each case if an elevated immunoglobulin index or oligoclonal bands has been detected in said test CSF sample, thereby obtaining an extended composite score.

[0020] The present invention may further comprise the above mentioned method, further comprising comparing said basic composite score or said extended composite score determined in said test CSF sample with a reference value.

[0021] The present invention may further comprise the above mentioned method, wherein if said basic composite score or said extended composite score is above the reference value, it is indicative for whether or not said subject suffers from multiple sclerosis.

[0022] The present invention may further comprise the above mentioned method, wherein the reference value is at least about 2.5.

[0023] Also envisage by the present invention is a method of determining an adjustment factor for the method of the present invention, comprising the steps of

a) determining

i) the ratio of the level of NK cells to the level of CD4⁺ T cells,

ii) the ratio of the level of B cells to the level of CD4⁺ T cells,

iii) the ratio of the level of CD8⁺ cells to the level of CD4⁺ T cells, and/or

iv) the ratio of the level of CD14⁺CD16 monocytes to the level of CD14⁺CD16⁺ monocytes;

in a reference group of cerebrospinal fluid (CSF) samples comprising at least one sample obtained from a subject having multiple sclerosis and at least one sample obtained from a healthy subject;

b) calculating a group average for any one or all of the ratios of step a i) - a iv) being determined in said reference group of CSF samples;

c) calculating the reciprocal value of each group average value of step b), thereby obtaining the adjustment factor.

[0024] Additionally, the method of determining an adjustment factor may comprise the ratio of the number of samples obtained from a subject having multiple sclerosis to the number of samples obtained from a healthy subject comprised in the reference group being between about 30 % to about 70 % and about 70 % to about 30 %.

[0025] Additionally, the method of determining an adjustment factor may comprise that the healthy subject has idiopathic intracranial hypertension (IIH).

[0026] The present invention also comprises a data processing system comprising a processor configured to perform a method comprising the steps of

a) obtaining

i) the detected level of NK cells,

ii) the detected level of CD4⁺ T cells,

iii) the detected level of B cells,

iv) the detected level of CD8⁺ cells,

v) the detected level of CD14⁺CD16 monocytes, and

vi) the detected level of CD14⁺CD16⁺ monocytes;

from a test CSF sample obtained from a subject;

b) determining

i) the ratio of the level of NK cells of step ai) to the level of CD4⁺ T cells of step aii), ii) the ratio of the level of B cells of step aiii) to the level of CD4⁺ T cells of step aii), iii) the ratio of the level of CD8⁺ cells of step aiv) to the level of CD4⁺ T cells of step aii), and iv) the ratio of the level of CD14⁺CD16 monocytes of step av) to the level of CD14⁺CD16⁺ monocytes of step avi);

C) multiplying

i) the ratio of the level of NK cells to the level of CD4⁺ T cells with an adjustment factor of preferably about 18 to about 74,

ii) the ratio of the detected level of B cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 25 to about 101 ,

iii) the ratio of the detected level of CD8⁺ cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 2 to about 8,

iv) the ratio of the detected level of CD14⁺CD16 monocytes to the detected level of CD14⁺CD16⁺ monocytes with an adjustment factor of preferably about 0.6 to about 2.3;

d) summing up each adjusted ratio of step c i) - c iv), thereby obtaining the basic composite score; optionally

e) adding a value of 1 to the basic composite score of step d) in each case if an elevated immunoglobulin index or oligoclonal bands has been detected in said test CSF sample, thereby obtaining an extended composite score; and

f) comparing said basic composite score or said extended composite score determined in said test CSF sample with a reference value; and

g) indicating whether or not said subject suffers from multiple sclerosis.

[0027] Also comprised by the present invention is a flow cytometry device capable of detecting i) the level of NK cells;

ii) the level of CD4⁺ T cells;

iii) the level of B cells;

iv) the level of CD8⁺ cells;

v) the level of CD14⁺CD16 monocytes;

vi) the level of CD14⁺CD16⁺ monocytes;

in a test CSF sample obtained from a subject, comprising the data processing system as defined above.

[0028] The present invention also envisages a computer program comprising instructions to cause the data processing system as defined above or the flow cytometry device as defined above to execute the steps of

a) obtaining

i) the detected level of NK cells,

ii) the detected level of CD4⁺ T cells,

iii) the detected level of B cells, iv) the detected level of CD8⁺ cells,

v) the detected level of CD14⁺CD16 monocytes, and

vi) the detected level of CD14⁺CD16⁺ monocytes,

from a test CSF sample obtained from a subject;

b) determining

i) the ratio of the level of NK cells of step ai) to the level of CD4⁺ T cells of step aii), ii) the ratio of the level of B cells of step aiii) to the level of CD4⁺ T cells of step aii), iii) the ratio of the level of CD8⁺ cells of step aiv) to the level of CD4⁺ T cells of step aii), and

iv) the ratio of the level of CD14⁺CD16 monocytes of step av) to the level of CD14⁺CD16⁺ monocytes of step avi);

c) multiplying

i) the ratio of the level of NK cells to the level of CD4⁺ T cells with an adjustment factor of preferably about 18 to about 74,

ii) the ratio of the detected level of B cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 25 to about 101 ,

iii) the ratio of the detected level of CD8⁺ cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 2 to about 8,

iv) the ratio of the detected level of CD14⁺CD16 monocytes to the detected level of CD14⁺CD16⁺ monocytes with an adjustment factor of preferably about 0.6 to about 2.3;

d) summing up each adjusted ratio of step c i) - c iv), thereby obtaining the basic composite score; optionally

e) adding a value of 1 to the basic composite score of step d) in each case if an elevated immunoglobulin index or oligoclonal bands has been detected in said test CSF sample, thereby obtaining an extended composite score; and

f) comparing said basic composite score or said extended composite score determined in said test CSF sample with a reference value; and

g) indicating whether or not said subject suffers from multiple sclerosis.

[0029] Also comprised by the present invention is a computer-readable medium having stored thereon the computer program as defined above.

[0030] The present invention further comprises a kit comprising a fluorescently labeled binding partner for CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and CD138, preferably wherein the binding partner is an immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like functions. [0031] Finally, the present invention further comprises an agent for use in the treatment of multiple sclerosis in a subject, wherein the subject has been diagnosed as having multiple sclerosis according to the method of the present invention, wherein the agent is selected from the group consisting of Interferon-beta 1a, Interferon-beta 1 b, Glatiramer acetate, Teriflunomide, Fingolimod, Dimethyl fumarate, Alemtuzumab, Ocrelizumab, Natalizumab, Cladribine, Daclizumab, Siponimod, Ofatumumab, high dose biotin, Mitoxantrone, Cyclophosphamide, Azathioprine.

BRIEF DESCRIPTION OF THE FIGURES

[0032] Fig. 1 : Single-cell transcriptomics reconstructs the CSF leukocyte composition.

(A) Schematic of the study. CSF cells of control (n = 22) and of multiple sclerosis (MS) (n = 26) donors were analysed by flow cytometry. scRNA-seq of unsorted CSF cells (n = 6 donors each group) and population RNA-seq of sorted T follicular helper (TFH) cells (n = 9 donors each group) were performed in randomly selected donors. (B) tSNE plot of 10 color-coded cell clusters identified by scRNA-seq after quality control and normalization (Methods) in 22,357 total merged control- (n = 4) and MS-derived (n = 4) CSF cells. Cluster identity was manually assigned based on marker gene expression. (C) The average proportion of cells in each cluster. Please note the split y-axis and higher magnification inset. (D) Feature plots of selected marker genes (selected corresponding protein names are provided for clarity) with expressing cell types indicated. (E) Violin plot of selected marker genes in clusters. (F) Average proportions of CSF leukocyte subsets identified by flow cytometry in full cohort. Be B cells, plasma plasma cells, class_mono / nc_mono classical / non-classical monocytes, mDC / pDC myeloid / plasmacytoid dendritic cells, nCD8 / aCD8 naive / activated CD8⁺ T cells, NK natural killer cells, Treg regulatory T helper cells, CD4 CD4⁺ T cells.

[0033] Fig. 2: Unbiased transcriptomics detects NK and B lineage CSF cell expansion in MS.

(A) tSNE plots of 10 colored-coded distinct cell clusters identified by scRNA-seq in control- (n = 4 donors, 12,705 cells, left plot) and MS-derived (n = 4 donors, 9,652 cells, right plot) CSF cells separated by disease-state. Plot orientation, colours, and labels correspond to Figure 1 B. (B) The average proportion of cells in each cluster in control and MS samples is depicted. Please note the split y-axis and higher magnification insets. (C) Vulcano plot of the mean fold change (log₂) against the significance p-value (log₁₀ adjusted linear model) of the change in cluster abundance in MS vs. control donors. Horizontal line indicates significance threshold (p < 0.01). (D) The CD4 cluster of cells depicted in Figure 1B was re-normalized and sub-clustered. tSNE plots of 8 CD4 cell sub-clusters identified in control- (n = 4 donors, 7,764 cells, left plot) and MS- derived (n = 4 donors, 6,749 cells, right plot) CSF cells. (E) Heatmap plotting mean expression level of marker genes (columns) against CD4 sub-cluster identity (rows) in the dataset depicted g

in panel D. (F) Average proportion of cells in each cluster. (G) Vulcano plot as in panel C of the change in CD4 sub-cluster abundance in MS vs. control donors. Horizontal lines indicate significance thresholds (blue p < 0.05, red p < 0.01 ) and inset highlights overlapping symbols. (H) The proportion of CSF leukocyte subsets identified by flow cytometry in control vs. MS donors is depicted. Be B cells, plasma plasma cells, class mono / ne mono classical / non- classical monocytes, mDC / pDC myeloid / plasmacytoid dendritic cells, nCD8 / aCD8 naive / activated CD8⁺ T cells, NK natural killer cells, Treg regulatory T helper cells, n_CD4 naive, prol_CD4 proliferating, cm_CD4 central memory, lem_CD4 late effector memory, eem_CD4 early effector memory, r-CD8 remaining CD8⁺ T cells, r-mono remaining monocytes.

[0034] Fig. 3: Late B lineage cells accumulate in the CSF in MS.

(A) Feature plot highlighting the expression level of different heavy chain transcripts in the B cell (Be) cluster identified in Figure 1 B. (B) Feature plot as in panel A showing expression of selected heavy and light chain transcripts in the plasma cell cluster. (C) Proportion of Be and plasma cells in control vs. MS in clustered scRNA-seq data. (D) Proportion of Be and plasma cells expressing the indicated heavy (left panel) and light (right panel) chain transcripts at maximum level.

[0035] Fig. 4: Deconvolution and interpretation of CSF cell transcriptomes and MS genetics.

(A) Available microarray data of unsorted CSF cells from controls and MS patients in relapse or remission were retrieved and cell type deconvolution was performed using cluster-specific gene expression (mean UMI counts) determined by scRNA-seq. Cluster names corresponding to Figures 1 and 2 are indicated above each plot. Significance was tested applying one-way ANOVA with Tukey's honestly significant differences. (B) Heatmap plotting expression (mean UMI counts) of 167 published MS risk genes (columns) against CSF cell cluster (rows). Columns were hierarchically clustered using One minus Pearson correlation and selected gene names are indicated. ^* p < 0.05, ^** p < 0.01 , ^*** p < 0.005

[0036] Fig. 5: Increased T follicular helper (TFH) cells in the CSF of MS patients.

(A) Representative flow cytometry dot plot of CSF cells from a control and MS patient stained for CD4 and CXCR5 after gating on live CD3⁺ cells. (B) The proportion of CXCR5⁺ (left), of PD-1⁺CXCR5⁺ (middle), and of ICOS⁺PD-1⁺CXCR5⁺ (right) cells among live CD3⁺CD4⁺ T cells in the CSF cells of control (co; n = 9) and MS (n = 9) patients was quantified by flow cytometry. ^* p < 0.05. (C) Correlation matrix of CXCR5⁺ populations and CD19⁺ and CD138⁺CD19⁺ B lineage cells in the CSF. Blue circles indicate significance and the Pearson correlation coefficient is indicated. (D) Live CD3⁺CD4⁺CXCR5⁺ cells quantified in panel B were flow sorted from the CSF of control (237±1Q7 SD cells) and MS patients (852±691 SD cells) bulk RNA-seq was performed. The log₂ of the mean fold expression change of genes with p < 0.1 between MS vs. control samples is depicted. Selected gene names are indicated.

[0037] Fig. 6: Patient characteristics.

(A) Clinical characteristics (age, sex) of all control (co, n = 22) and multiple sclerosis (MS, n = 26) patients included into the study after screening are depicted. Recruitment into the study cohorts is depicted. (B) MS patients were classified to either have (Gd+) or not have (no Gd) contrast enhancing lesions in brain or spinal cord by magnetic resonce imaging. Oligoclonal bands (OCB) in CSF were classified as being either undetectable (type 1 ), or restricted to CSF (type 2), or detected in serum and additionally in CSF (type 3), or not determined (?). CSF/serum indices for albumin and immunoglobulin G (IgG) were calculated. The CSF barrier function was evaluated as being either unaffected (none), or showing intrathecal IgG synthesis (Igonly), or showing barrier dysfunction (barrier only), or showing both intrathecal IgG synthesis and barrier dysfunction (barrier_and_lg). (C) Standard CSF parameters of all study patients including concentrations in CSF of total cells, granulocytes, red blood cells (RBC), protein, lactate, glucose were quantified. (D) The study recruitment scheme is depicted. 53% of control and 35% of MS samples were excluded after screening for the reasons indicated. (E) Clinical characteristics (age, sex) of patients excluded after screening and reasons for exclusion are shown. NA not applicable.

[0038] Fig. 7: Inter- and intra-method correlation of scRNA-seq and flow cytometry results.

(A) Correlation matrix of cell numbers per cluster in the CSF scRNA-seq data. Light grey and black circles indicate negative and positive correlation, respectively and circle size represents the absolute value of the correlation coefficient. (B) Correlation matrix of CSF flow cytometry parameters assessed in the study. (C) Correlation matrix of flow cytometry vs. scRNA-seq data. Cluster names correspond to Figure 1.

[0039] Fig 8: Flow cytometry characterization of all CSF cell samples.

(A) Representative gating strategy for identifying and quantifying cell types by flow cytometry in the CSF. Population names are indicated next to the respective gates. (B) Quantification of the indicated cell types in CSF in control (co) and MS patients. All percentages are expressed as proportion of CD45⁺ cells. Samples with less than 500 total CD45⁺ events analysed by flow cytometry were excluded from NK quantification. (C) Circle plot depicting the proportion of cell types among all CD45+ cells in the CSF in control and MS. (D) Donor-specific proportions of cells in each cluster identified by scRNA-seq (note split y-axis) and (E) in each CD4+ T-cell (CD4_Tc) subcluster, for all control and MS patients individually. Cluster key: CD4_Tc: CD4+ T cells, aCD8_Tc / nCD8_Tc: activated / naive CD8+ T cells, NK: natural killer cells, Be: B cells, plasma: plasma cells, class mono / nc mono: classical / non-classical monocytes, mDC / pDC: myeloid / plasmacytoid dendritic cells; Treg: regulatory T helper cells, n CD4: naive, prol_CD4: proliferating, cm_CD4: central memory, lem_CD4: late effector memory, eem_CD4: early effector memory, r-CD8: remainingCD8+ T cells, r-mono: remaining monocytes.

[0040] Fig. 9: Evaluating a composite score for diagnosing MS by CSF analysis.

(A) In the merged flow cytometry data depicted in Fig. 8, for each sample a normalized ratio of the proportion of NK to CD4⁺ T cells, of B to CD4⁺ T cells, of CD8⁺ to CD4⁺ T cells, and of CD14⁺CD16⁺ to CD14⁺CD16⁺ monocytes have been calculated. These four normalized ratios were added to a composite score that is depicted in control (co) vs. MS samples. (B) Receiver operator curve (ROC) analysis plotting sensitivity against 1 -specificity and the area under the curve (AUC) of the composite. (C) Values of 1 were added to the basic composite depicted in panel A if an elevated immunoglobulin index or oligoclonal bands were detected in the sample. This extended composite score values are depicted by disease status. (D) Receiver operator curve (ROC) analysis of extended composite score.

[0041] Fig. 10: ROC analysis with multiple composite scores.

(A) ROC analysis with the basic composite score based on the ratio of NK cells:CD4+ T cells, B cells:CD4+ T cells, CD8+:CD4+ T cells, activated : non-activated T cells and classical:non- classical monocytes, being calculated by addition of each adjusted ratio, AUC: 0.8588. (B) ROC analysis with the basic composite score based on the ratio of NK cells:CD4+ T cells, B cells:CD4+ T cells, CD8+:CD4+ T cells and classical: non-classical monocytes, being calculated by addition of each adjusted ratio, AUC: 0.8618. (C) ROC analysis with the extended composite score based on the ratio of NK cells:CD4+ T cells, B cells:CD4+ T cells, CD8+:CD4+ T cells and classicahnon-classical monocytes and based on the determination of an elevated immunoglobulin index and oligoclonal bands (OCB), being calculated by addition of each adjusted ratio and adding a value of 2 to said basic composite score since an elevated IG index and oligoclonal bands have been detected in the CSF sample as well, AUC: 0.9176. (D) ROC analysis with the basic composite score based on the ratio of B cells:CD4+ T cells and classical:non-classical monocytes, being calculated by addition of each adjusted ratio, AUC: 0.8324. (E) ROC analysis with the basic composite score based on the ratio of NK cells:CD4+ T cells, B cells:CD4+ T cells, CD8+:CD4+ T cells, activated: non-activated T cells and classical:non-classical monocytes, being calculated by multiplication of each adjusted ratio, AUC: 0.8118. (F) ROC analysis with the basic composite score based on the ratio of NK cells:CD4+ T cells, B cells:CD4+ T cells, CD8+:CD4+ T cells and classical: non-classical monocytes, being calculated by multiplication of each adjusted ratio, AUC: 0.8147. (G) ROC analysis with the extended composite score based on the ratio of NK cells:CD4+ T cells, B cells:CD4+ T cells, CD8+:CD4+ T cells and classicahnon-classical monocytes and based on the determination of an elevated immunoglobulin index and oligoclonal bands (OCB), being calculated by multiplication of each adjusted ratio and adding a value of 2 to said basic composite score since an elevated IG index and oligoclonal bands have been detected in the CSF sample as well, AUC: 0.7. (H) ROC analysis with the basic composite score based on the ratio of B cells:CD4+ T cells and classical:non-classical monocytes, being calculated by multiplication of each adjusted ratio, AUC: 0.8088.

[0042] Fig. 11 : Calculation of four different composite scores of treatment-na^'fve patients with relapsing-remitting multiple sclerosis in comparison to patients suffering from idiopathic intracranial hypertension as a control. Treatment-na^'fve patients with relapsing- remitting multiple sclerosis (RRMS, n = 49) and patients suffering from idiopathic intracranial hypertension (IIH, n = 52) as a control were retrospectively identified. All patients had flow cytometry data of cerebrospinal fluid (CSF) cells and standard CSF parameters (e.g. CSF protein) available. The following parameters from the CSF parameter database were then extracted (see the abbreviations for these parameters provided in brackets): 1 ) presence of intrathecal immunoglobulin synthesis as determined by an increased immunoglobulin index (index), 2) presence of oligoclonal bands (ocb), 3) percentage of B cells (B), 4) percentage of natural killer cells (NK), 5) ratio between CD4+ T cells and CD8+ T cells (8to4), 6) percentage of activated T cells (act), 7) percentage of non-classical monocytes (nc). Four different composite scores were then obtained by calculating the sum of different single parameters after normalization: composite score 1 : NK+B+8to4+act+nc, composite score 2: NK+B+8to4+nc, Composite 3: B+nc, composite score 4: NK+B+8to4+ nc+ocb+index. The numerical values of the individual composite score were then plotted. Box and whiskers indicate mean ± SEM.

[0043] Fig. 12: ROC analysis of the extended composite score (NK+B+8to4+nc+ocb+index). A receiver operator curve (ROC) analysis with the four composite scores outlined in detail in Figure 11 has been performed: composite score 1 : NK+B+8to4+act+nc, composite score 2: NK+B+8to4+nc, composite score 3: B+nc, composite score 4: NK+B+8to4+nc+ocb+index. The ROC analysis of the composite score 1 : NK+B+8to4+act+nc and the extended composite score 4: NK+B+8to4+nc+ocb+index which returned highest area under the curve (AUC) values in the ROC analysis are depicted herein. DETAILED DESCRIPTION OF THE INVENTION

[0044] Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0045] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments described throughout the specification should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all elements described herein should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0046] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps although in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e. the subject-matter consists in the inclusion of a stated member, integer or step or group of members, integers or steps. When used herein the term“comprising” can be substituted with the term“containing” or“including" or sometimes when used herein with the term“having”. When used herein“consisting of excludes any element, step, or ingredient not specified.

[0047] The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. [0048] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0049] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. The term“at least one” refers to one or more such as two, three, four, five, six, seven, eight, nine, ten and more. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0050] The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0051] When used herein“consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0052] The term“including” means“including but not limited to”.“Including” and“including but not limited to” are used interchangeably.

[0053] The term “about” means plus or minus 20%, preferably plus or minus 10%, more preferably plur or minus 5%, most preferably plus or minus 1 %.

[0054] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0055] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0056] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0057] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

[0058] A better understanding of the present invention and of its advantages will be gained from the examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.

Method

[0059] As used herein, the method of the present invention may be applied to diagnose a subject with multiple sclerosis.

[0060] The term “multiple sclerosis (MS)” as used herein refers to a chronic, inflammatory central nervous system (CNS) disease, characterized pathologically by demyelination. MS has also been classified as an autoimmune disease. It refers to a demyelinating disease in which the insulating covers of nerve cells in the brain and spinal cord are damaged. MS disease activity can be monitored by cranial scans, including magnetic resonance imaging (MRI) of the brain, accumulation of disability, as well as rate and severity of relapses. There are five distinct disease stages and/or types of MS, namely, (1 ) clinically isolated syndrome (CIS); (2) relapsing- remitting multiple sclerosis (RRMS); (3) secondary progressive multiple sclerosis (SPMS); (4) progressive relapsing multiple sclerosis (PRMS); and (5) primary progressive multiple sclerosis (PPMS). However, CIS is a pre-stage / pre-type of MS. When a subject is diagnosed with MS by the method of the present invention, said subject may be diagnosed with any one of the five disease stages or types of MS mentioned above. By forgoing differential diagnosis of the prior art, which are very laborious, the present invention however demonstrates a new method of diagnosing said disease in a subject by determining

i) the ratio of the level of NK cells to the level of CD4⁺ T cells;

ii) the ratio of the level of B cells to the level of CD4⁺ T cells;

iii) the ratio of the level of CD8⁺ cells to the level of CD4⁺ T cells, and

iv) the ratio of the level of CD14⁺CD16 monocytes to the level of CD14⁺CD16⁺ monocytes;

in a test cerebrospinal fluid (CSF) sample obtained from said subject, wherein the combination of said ratios as defined in i) - iv) is indicative for whether or not said subject suffers from multiple sclerosis.

[0061] The term“diagnose / diagnosing” as used herein and through the entire description refers to making a diagnosis that a subject will suffer from multiple sclerosis (from any one of the five defined disease stages or types of MS) in the near future or confirming a diagnosis that a subject suffers indeed from multiple sclerosis, which has been suspected to suffer from any one of the five defined disease stages or types of MS, before said method of the present invention has been applied to said subject.

[0062] The term“to have / having MS” can be used interchangeably with the term“to suffer from / suffering from MS”. In general, when a subject suffers from a disease, said subject shows specific symptoms of the disease, whereas when a subject has a disease, said subject does not always have to show certain symptoms of the disease, but still is diagnosed with said disease. However, this general concept does not apply to MS.

[0063] The term“subject” as used herein and throughout the entire description, also addressed as an individual, refers to a human or non-human animal, generally a mammal. A subject may be a mammalian species such as a rabbit, a mouse, a rat, a Guinea pig, a hamster, a dog, a cat, a pig, a cow, a goat, a sheep, a horse, a monkey, an ape or a human. Preferably, the subject being used in the present invention is a human. More preferably, said subject is an adult. Preferably, said adult is older than 18 years. More preferably, said adult is about 20 to 50 years, about 25 to 45 years, about 30 to 40 years, about 25 years, about 28 years, about 30 years, about 32 years old.

[0064] In a preferred embodiment, said subject is suspected to suffer from (any one of the five disease stages of) MS. Thus, said subject may also be suspected to suffer from the pre-stage of MS termed as CIS. CIS refers to a first episode suggestive of MS. It is a first episode of neurologic symptoms that lasts at least about 24 hours and is caused by inflammation or demyelination (loss of the myelin that covers the nerve cells) in the central nervous system (CNS). Some reviews describe CIS as "the prodromal stage of MS” (Dupont et al. 2017, International Journal of Molecular Sciences. 18 (4): 785), an early sign or symptom (or set of signs and symptoms), which often indicate the onset of a disease before more diagnostically specific signs and symptoms develop. Dissemination in time (DIT) is what distinguishes CIS from RRMS. If a subject may be examined with a first demyelinating event and dissemination in space (DIS) on an MRI scan, said subject may still have / suffer from CIS, since DIT criteria have not been fulfilled. As an illustrative example for revision of MS diagnostic criteria see the scenarios below:

Scenario (A) first event and exclusion of differential diagnoses: if no DIS may be examined, the subject may then be diagnosed with CIS or none of the other four stages of MS;

Scenario (B) first event and exclusion of differential diagnoses: if DIS, but no DIT may be examined, the subject may then be diagnosed with CIS;

Scenario (C) First event and exclusion of differential diagnoses: if DIS and DIT may be examined, the subject may then be diagnosed with RRMS;

Scenario (D) Many years of RRMS, but now slowly progressing, the subject may then be diagnosed with SPMS;

Scenario (E) exclusion of differential diagnoses: if relapses have never occurred, but slowly progressing from the start, the subject may then be diagnosed with PPMS.

[0065] The term“suspected to suffer” refers to assuming that said subject being examined by using the method of the present invention might suffer from MS (from any one of the five defined disease stages or types of MS) based on the general diagnosing test(s) available in the prior art (e.g., MRI), which has/have already been applied to said subject for the diagnosis before the method of the present invention has been applied as a confirmation procedure.

[0066] According to the present invention, it may also be comprised that a subject not being diagnosed with MS based on the general diagnosing test(s) available in the prior art (e.g., MRI), which has/have been applied to said subject, may then be diagnosed with MS (with any one of the five disease stages or types of MS) by applying the method of the present invention. Thus, the method of the present invention may also diagnose a specific patient group, which has not been diagnosed with MS or which failed to be diagnosed with MS by applying classical MS diagnosing procedures known to a person skilled in the art.

[0067] Determining each ratio of the level of specific cells to another level of other specific cells as defined above means comparing the level of specific cells to the level of other specific cells (e.g. comparing the level of NK cells to the level of CD4⁺ T cells), or putting said levels of cells into relation to one another. Thus, determining the ratio of the level of NK cells in relation to the level of CD4⁺ T cells may also be comprised herein. This wording also applies mutatis mutandis to the other cell ratios as defined above as well.

[0068] Thus, the term“in relation to" or just“to” as used herein in this context, e.g.“the level of A” (in relation) to“the level of B”, generally means comparing “A” to“B”. Preferably, this comparison is carried out by dividing A by B, thereby obtaining a ratio value. Here, the relation of“A” to“B” can be expressed as the quotient of“A” divided by“B”, again thereby obtaining a ratio value. As an illustrative example,“the level of NK cells" (in relation) to the“level of CD4⁺ T cells” may be expressed by the quotient when dividing“the level of NK cells” by the“level of CD4⁺ T cells” (or dividing the“level of B cells” by the“level of CD4⁺ T cells”; dividing“the level CD8⁺ T” cells by“the level of CD4⁺ T cells”; dividing“the level of CD14⁺ CD16- monocytes” by “the level of CD14⁺ CD16⁺ monocytes”). For example the level of NK cells in relation to the level of CD4+ T cells determined in or from a sample of a subject may be expressed in terms of a decimal value or a percentage, i.e. the quotient of dividing the level of NK cells by the level of CD4+ T cells. Therefore, determining the ratio of“A” (e.g., the detected level of NK cells) to / in relation to“B” (the detected level of CD4+ T cells) means calculating the specific ratios by dividing“A” (e.g., the detected level of NK cells) by the“B” (the detected level of CD4+ T cells). This wording also applies mutatis mutandis to the other cell ratios as defined above as well.

[0069] It is further encompassed by the present invention that the level of NK cells, the level of CD4+ T cells, the level of B cells, the level of CD8+ T cells, the level of CD14+CD16- monocytes and the level of CD14+CD16+ monocytes are detected using flow cytometry.

[0070] Flow cytometry based analysis is typically combined with optical detection to identify and classify cells. This allows speed, sensitivity/specificity, and a non-invasive nature of the technique. Typically fluorescent markers are used, which are compounds that bind to specific structures or molecules on the surface or within target cells. Such fluorescent markers are introduced into the mixture of cells, whereafter the mixture is rinsed to remove excess fluorescent markers. It is envisaged that flow cytometry may be combined with immunofluorescence. It is thus contemplated by the invention that the level of each specific cells defined above may be detected by detecting the specific surface molecules on the surface of each cells by using flow cytometry being combined with immunofluorescence. In the present invention CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56 and/or CD138 may be detected on the surface of the specific cells in the sample using a flow cytometry based analysis.

[0071] The term“detect” or“detecting”, as well as the term“determine” or“determining” when used herein in combination with the words“level”, “amount” or“value”, the words“detect”, “detecting”,“determine” or“determining” are understood to generally refer to a quantitative or a qualitative level. For example, when used in the context of detecting the level of a certain cell population, such as CD4+ T cells “detect”, “detecting”, “determine” or “determining” are understood to generally refer to a quantitative level. Accordingly, methods according to the invention that include a quantification of CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 - i.e. the amount or number of CD45expressing, CD3expressing, CD4expressing, CD8expressing, CD14expressing, CD16expressing, CD19expressing, CD56expressing, and/or CD138 expressing cells. In this regard the words “value,” “amount" and “level” are used interchangeably herein. The invention involves detection of the“level”, i.e. number or (relative) amount of CD4+ T cells. The level of specific cells may be expressed by the amount of specific cells being detected. It can also be expressed by the strength of a signal measured in the method of detecting the level of specific cells when immunofluorescence may be used, which may be combined with flow cytometry.

[0072] Generally, the invention envisages that the detection of the biomarkers CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 can be carried out in a single step or can be carried out in more than one step, e.g. two steps, three steps or four steps. In preferred embodiments, the detection is carried out in a single step. A preferred method for the detection of said specific cell levels as defined above is FACS. The skilled person in the art knows that if detection of the biomarkers is to be carried out in more than one step, more than one sequences of steps can be selected. It is understood that the skilled person is able to recognize suitable sequences of steps for identifying the cell populations of interest. Also, if more than one step is carried out, the steps may partly involve enriching and/or isolating subpopulations. Again, the skilled person is able to recognize suitable sequences of steps and can judge whether those steps would reasonably involve an enrichment and/or isolation step for identifying the cell populations of interest.

[0073] Flow cytometry is a technique for counting, examining, and sorting microscopic particles such as biological cells suspended in a stream of fluid. It allows a simultaneous multi-parametric analysis of the physical and chemical characteristics of single cells flowing through an optical or electronic detection device. An illustrative example of a well-established flow cytometry based analysis in the art is FACS. FACS allows sorting a heterogeneous mixture of cells into a plurality of containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. Thereby FACS allows the sorting of subpopulations of cells of interest and their further use in in vitro and in vivo assays. FACS is often used in combination with monoclonal immunoglobulins as a reagent to detect cells as having a particular antigen, indicative of an expressed protein.

[0074] This technique allows the concurrent fast, objective and quantitative recording of fluorescent signals from individual cells and the physical separation of respective cells according to particular interest. Fluorescent signals used in flow cytometry, for instance when quantifying and/or sorting cells by any marker present on or in the cell, are typically fluorescently-tagged antibody preparations or fluorescently-tagged ligands for binding to antibodies or other antigen-, epitope- or ligand-specific agent, such as with biotin/avidin binding systems or fluorescently-labeled and optionally addressable beads (e.g. LUMINEX® microspheres). Depending of the equipment used, any desired detectable marker or combination of detectable markers can be detected by the optics and/or electronics of a flow cytometer. Current three-laser, "multidimensional", FACS machines enable up to 14 simultaneous single-cell measurements, such as two light scatter detectors and 12 fluorescence plus forward detectors allowing for example the detection of fluorescent surface/intracellular markers. As an illustrative example, the three lasers of a FACS machine may be a krypton laser operating at 407 nm, an argon laser operating at 488 nm, and a dye laser operating at 595 nm.

[0075] The FACS technique has been used extensively in relation to antigens expressed on the surface of cells, including cells that remain alive during, and after, FACS. Similarly, the method has been used with intracellular reporter gene systems based on the expression of a detectably labeled gene product by the cell. Accordingly, the technique not only allows detecting the presence of CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 on the cell surface, but also detecting the presence of RNA or DNA within the cell, for example RNA encoding CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138. Therefore FACS can also be used to determine the amount of nucleic acid formation from the genes, which encode CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 in cells of the sample from the subject. In preferred embodiments, FACS technique is used for detecting the level of NK cells, the level of CD4+ T cells, the level of B cells, the level of CD8+ T cells, the level of CD14+CD16- monocytes and the level of CD14+CD16+ monocytes, preferably by detecting each specific cell surface marker / molecule such as CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 on the cell surface of each specific cells, which demonstrates a certain strength of a signal being measured.

[0076] The present invention contemplates that the presence of the CD45 on the surface of a cell may be used to identify the cell as a leukocyte.

CD45 (Cluster of Differentiation 45 or leukocyte common antigen) protein is a member of the protein tyrosine phosphatase (PTP) family and is expressed by all leukocytes. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. This PTP contains an extracellular domain, a single transmembrane segment and two tandem intracytoplasmic catalytic domains, and thus belongs to receptor type PTP. CD45 is a type I transmembrane protein that is in various forms present on all differentiated hematopoietic cells, except erythrocytes and plasma cells that assists in the activation of those cells (a form of co-stimulation). The CD45 family consists of multiple members that are all products of a single complex gene. This gene contains 34 exons and three exons of the primary transcripts are alternatively spliced to generate up to eight different mature mRNAs and after translation eight different protein products. These three exons generate the RA, RB and RC isoforms. Various isoforms of CD45 exist: CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, CD45R (ABC). CD45RA is located on naive T cells and CD45RO is located on memory T cells. CD45 is also highly glycosylated.

[0077] The invention also contemplates that the presence of the FcyRIII (CD16) and/or NCAM (neural cell adhesion molecule, also called CD56) on the surface of a cell may be used to identify the cell as a natural killer cell (NK cell). Thus, detecting the level of NK cells may comprise measuring CD45, CD16 and/or CD56. NK cells are a type of cytotoxic lymphocytes providing rapid responses to viral-infected cells and respond to tumor formation for example. They do not express T-cell antigen receptors (TCR) or pan T marker CD3, but they express the surface marker CD16 and CD56 in humans. According to the invention, identifying CD16+CD56+ cells in the sample serves in distinguishing CD16+ CD56+ cells from other cells such as CD16- CD56- cells. Antibodies that bind to antigens can be recognised by FcyRIII (CD16) receptors expressed on NK cells, resulting in NK activation, release of cytolytic granules and consequent cell apoptosis. CD16 may also be found not only on the surface of NK cells, but also on neutrophil polymorphonuclear leukocytes, monocytes and macrophages. Therefore, NK cells need to be distinguished from for example monocytes also expressing CD16 by another surface molecule such as CD56, which is most stringently associated with NK cells. CD16 has been identified as Fc receptors FcyRIIIa (CD16a) and FcyRIIIb (CD16b), which participate in signal transduction. While FcyRIIIa is expressed on mast cells, macrophages, and natural killer cells as a transmembrane receptor, FcyRIIIb is only expressed on neutrophils.

[0078] Additionally, the invention contemplates that the presence of CD3 (Cluster of Differentiation 3) and/or CD4 (Cluster of Differentiation 4) on the surface of a cell may be used to identify the cell as CD4+ T cell. Thus, detecting the level of CD4⁺ T cells may comprise measuring CD45, CD3 and/or CD4.

In general, T cells are known to the skilled artisan as lymphocytes, i.e. nucleated blood cells that are also called white blood cells. T cells mature in the thymus and can be distinguished from other lymphocytes in that they have the T cell receptor on their cell surface. The main known role of the T cell is recognition of antigens bound to major histocompatibility complex (MHC) molecules. The T cell receptor (TCR) is a heterodimer, which consists of a 34 kD a- chain, linked by a disulphide bond to a 34 kD b-chain in about 95 % of T cells. Both chains span the plasma membrane and have accordingly an extracellular portion, each of which includes a variable region, termed Va and V b, respectively. About 5 % of T cells have a T cell receptor that consists of a y- and a d-chain instead of an a - and a b-chain, which likewise have extracellular variable regions. T cell receptors can, like immunoglobulins, recognize a very large number of different epitopes. As the T cell receptor has variable regions it may, nevertheless, be advantageous to use another cell surface protein to identify a T cell. An example of suitable protein in this regard is a T cell co-receptor. An illustrative examples of a co-receptor of the T cell receptor is the protein complex CD3 (Cluster of Differentiation 3). CD3 has four chains, which are in mammals one CD3y chain (e.g. human CD3y of the UniProt accession number P09693, version 147 of 7 January 2015 or corresponding mRNA of the GenBank Accession number X04145, version X04145.1 Gl:37021 of 18 April 2005), one CD36 chain (e.g. human CD36 of the UniProt accession number P04234, version 157 of 7 January 2015 or corresponding mRNA of the GenBank Accession number BC039035, version BC039035.1 Gl:25058311 of 19 June 2006), and two CD3E chains (e.g. human CD3E of the UniProt accession number P07766, version 167 of 7 January 2015 or corresponding mRNA of the GenBank Accession number X03884, version X03884.1 Gl:37039 of 07 April 1994). These chains associate with a molecule known as the T-cell receptor and at least one T-cell surface glycoprotein CD3 zeta chain also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247) (e.g. human CD3 zeta chain of the UniProt accession number P20963, version 165 of 7 January 2015 or corresponding mRNA of the GenBank Accession number J04132, version J04132.1 Gl:623041 of 12 January 1995). The complex of TCR, CD247 and CD3 can generate an activation signal in T lymphocytes. The TCR, z-oIΐ3ίh(5), and CD3 molecule together define the TCR complex. In practicing the methods according to the invention identifying the presence of CD3 on a particular cell or plurality of cells is often a convenient way of identifying T cells. Therefore, the terms“CD3+ T cell” and“T cell” are used interchangeable herein to address a T cell and to distinguish a T cell from other cell types.

According to the invention, identifying CD3+ T cells in the sample serves in distinguishing CD3+ T cells from other cells such as CD3- cells or non-T cells (such as NK cells and/or B cells).

In the methods according to the invention, the level of T cells in the sample that is CD4 positive (CD4+) are detected. Identifying CD4+ T cells typically serves in distinguishing CD4+ T cells from other cells such as CD4- T cells. CD4+ T cells in addition to CD3 have the CD4 (Cluster of Differentiation 4) protein on their surface, a glycoprotein consisting of four extracellular immunoglobulin domains, termed D1 to D4, and a small cytoplasmic region (e.g. human CD4 of the UniProt accession number P01730, version 180 of 7 January 2015 or corresponding mRNA of the GenBank Accession number M12807, version M12807.1 Gl:179141 of 27 April 1993). CD4+ T cells can be classified into a variety of cell populations with different functions and should thus not be taken to define a unitary set of cells. Typical examples of a CD4+ T cell are naive CD4+ T-cells (naive),“central” memory CD4+ T-cells (TCM), or“effector” memory CD4+ T cells (TEM).

[0079] In the method according to the invention, the level of T cells in the sample being CD8 positive (CD8+) may also be detected. Thus, detecting the level of CD8⁺ T cells may comprise measuring CD45, CD3 and/or CD8. Identifying CD8+ T cells typically serves in distinguishing CD8+ T cells from other cells such as CD8- T cells. CD8+ T cells in addition to CD3 have the CD8 (Cluster of Differentiation 8) protein on their surface, a transmembrane glycoprotein consisting of a pair of CD8 chains, most commonly composed of a CD8- a and CD8- b chain having a molecular weight of about 34 kDa. The CD8 co-receptor is predominantly expressed on the surface of cytotoxic T cells. Like the TCR, CD8 interacts with the MHC molecule, but is specific for the Class I MHC molecule (in particular it interacts with a₃ portion of the Class I MHC molecule). [0080] Additionally, the invention contemplates that the presence of CD19 (Cluster of Differentiation 19) and/or CD 138 (Cluster of Differentiation 138) on the surface of a cell may be used to identify the cell as a B cell. Thus, detecting the level of B cells may comprise measuring CD45, CD19 and/or CD138. B cells are also a type of white blood cell (leukocytes) of the lymphocyte subtype. They function in the adaptive immune system by secreting antibodies. Also B cells present antigens and secrete cytokines. They express B cell receptors (BCRs) on their cell surface, thereby allowing the B cell to bind specific antigens, against which the B cell will initiate an antibody response.

[0081] According to the invention, identifying CD19+ cells in the sample serves in distinguishing CD19+ cells from other cells such as CD19- cells (such as monocytes, T cells, NK cells). CD19 also known as B-lymphocyte antigen CD19 is a transmembrane protein encoded in humans by the gene CD19 and is a biomarker for B lymphocyte development being widely expressed during all phases of B cell development until terminal differentiation into plasma cells. CD19 plays two major roles in human B cells: 1 ) acting as an adaptor protein to recruit cytoplasmic signaling proteins to the membrane; 2.) working within the CD19/CD21 complex to decrease the threshold for B cell receptor signaling pathways.

Further, identifying CD19+ CD138+ cells refer to B cells, also expressing CD138 (syndecan-1 ) which is a transmembrane proteoglycan preferably with a main cellular expression in stratified and simple epithelia and which is mainly confined to late stages of B-cell differentiation.

[0082] Also the invention contemplates that the presence of CD14 (Cluster of Differentiation 14) and/or CD 16 (Cluster of Differentiation 16) on the surface of a cell may be used to identify the cell as a monocyte. Thus, detecting the level of monocytes may comprise measuring CD45, CD14 and/or CD16.

As mentioned above, CD16 may also be expressed not only on monocytes, but also on NK cells. Thus, another surface molecule may be determined to detect the level of monocytes, such as CD14. Monocytes represent a heterogeneous population of primary immune effector cells. They are the largest type of leukocyte and can differentiate into macrophages and myeloid lineage dendritic cells. There are three different subsets being distinguished based on their expression of CD14 and the low-affinity CD16 (FcyRIII): CD14++CD16- classical monocytes, which are characterized by high level expression of the CD14 cell surface receptor. CD14++CD16+ intermediate monocytes, which are characterized by high level expression of CD14 and low level expression of CD16 and CD14+CD16++ non-classical monocytes, which are characterized by low level expression of CD14 and high co-expression of the CD16 receptor.

According to the present invention, CD14+CD16- monocytes refer to classical monocytes as defined above, whereas CD14+CD16+ refer to non-classical monocytes having a high expression level of CD16.

[0083] The term“measuring a surface marker / molecule (e.g., CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56 and/or CD138)” refers to measuring the signal of a detectable marker, which is attached to a binding partner that recognizes the specific surface marker / molecule expressed by the specific cell when using flow cytometry. Said signal may then be converted by the process called gating, which is known to a person skilled in the art, which then demonstrates the level (or relative amount) of cells being detected in said sample.

[0084] The measurement used is generally selected to be of a sensitivity of detection that allows detection of CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 expressing cells in the range of a selected threshold value, in particular of a sensitivity of detection that allows determining whether CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 expressing cells are above the threshold.

[0085] It is understood that if detection is carried out using flow cytometry or FACS, a binding partner of CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 may be used, preferably linked to excitable fluorescent dyes or proteins. Such a binding partner of CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 has a detectable affinity and specificity for CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138. Typically, binding is considered specific when the binding affinity is higher than 10 ⁶ M. A binding partner of CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138, has in some embodiments an affinity of about 10⁸ M or higher, or of about 10⁹ M or higher. As indicated above, in some embodiments for example T cells in the sample are identified by the presence of the CD3 protein on their surface; or T cells may be enriched or isolated via the presence of the CD3 protein on their surface. Identification of CD3+ T cells may be carried out using spectroscopic, photochemical, photometric, fluorometric, radiological, enzymatic or thermodynamic means. Identification and enrichment or isolation of T cells may likewise be carried out by using a suitable binding partner of CD3+. Accordingly the above said applies mutatis mutandis to identifying and enriching or isolating NK cells, B cells, CD4+ T cells, CD8+ T cells, classical and non-classical monocytes by using a suitable binding partner of their specific surface molecules.

[0086] Further, in some embodiments for example T cells may be identified or isolated in a similar manner, using suitable surface proteins known in the art, for example the T cell receptor. In some embodiments a suitable binding partner of CD3 and a further suitable binding partner of a surface protein characteristic for T cells such as the T cell receptor are combined to identify CD3+ T cells. Accordingly the above said applies mutatis mutandis to identifying and enriching or isolating NK cells, B cells, CD4+ T cells, CD8+ T cells, classical and non-classical monocytes by using a suitable binding partner of their specific surface molecules and a further suitable binding partner of a surface protein characteristic for said cells.

[0087] Immunofluorescence being used in flow cytometry is generally achieved using a binding partner, which is linked to, or includes, a fluorophore as a detectable marker. Typically a binding partner of CD3 may be used in combination with a detectable marker or the binding partner is functionally linked to a detectable marker. Likewise a binding partner of CD45, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 may be used in combination with a detectable marker or the binding partner is functionally linked to a detectable marker.

[0088] In some embodiments a suitable binding partner of CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 are combined to identify cells expressing a combination of biomarkers selected from CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138. In an illustrative example, a suitable binding partner of CD45, a suitable binding partner of CD16, and a suitable binding partner of CD56 are combined to identify NK cells. In another illustrative example, a suitable binding partner of CD45, a suitable binding partner of CD3, and a suitable binding partner of CD4 are combined to identify CD4+ T cells. In a further illustrative example, a suitable binding partner of CD45, a suitable binding partner of CD19, and a suitable binding partner of CD138 are combined to identify B cells. In another illustrative example, a suitable binding partner of CD45, a suitable binding partner of CD3, and a suitable binding partner of CD8 are combined to identify CD8+ T cells. In another illustrative example, a suitable binding partner of CD45, a suitable binding partner of CD14, and a suitable binding partner of CD16 are combined to identify classical monocytes and/or non-classical monocytes. The above mentioned binding partner may be functionally linked to a detectable marker.

[0089] A respective binding partner of e.g. CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 as well as a binding partner for another selected cell-characteristic protein, may be an immunoglobulin, a fragment thereof or a proteinaceous binding molecule with immunoglobulin-like functions. An antibody fragment generally contains an antigen binding or variable region. Examples of (recombinant) antibody fragments are immunoglobulin fragments such as Fab fragments, Fab’ fragments, Fv fragments, single-chain Fv fragments (scFv), diabodies or domain antibodies (Holt, L.J., et al., Trends Biotechnol. (2003), 21 , 1 1 , 484-490). An example of a proteinaceous binding molecule with immunoglobulin-like functions is a mutein based on a polypeptide of the lipocalin family (WO 03/029462, Beste et al., Proc Nat. Acad Sci 1999; 96:1898-1903). Lipocalins, such as the bilin binding protein, the human neutrophil gelatinase-associated lipocalin, human Apolipoprotein D or glycodelin, possess natural ligand- binding sites that can be modified so that they bind to selected small protein regions known as haptens. Examples of other proteinaceous binding molecules are the so-called glubodies (see e.g. international patent application WO 96/23879 or Napolitano et al., Chemistry & Biology 1996; 3(5):359-367), proteins based on the ankyrin scaffold (Mosavi et al., Protein Science 2004; 13(6): 1435-1448) or crystalline scaffold (e.g. internation patent application WO 01/04144), the proteins described in Skerra, J. Mol. Recognit. 2000; 13:167-187, AdNectins, tetranectins and avimers. Avimers contain so called A-domains that occur as strings of multiple domains in several cell surface receptors (Silverman et al., Nature Biotechnology 2005; 23:1556-1561 ). Adnectins, derived from a domain of human fibronectin, contain three loops that can be engineered for immunoglobulin-like binding to targets (Gill & Damle, Current Opinion in Biotechnology 2006; 17:653-658). Tetranectins, derived from the respective human homotrimeric protein, likewise contain loop regions in a C-type lectin domain that can be engineered for desired binding. Peptoids, which can act as protein ligands, are oligo(N-alkyl) glycines that differ from peptides in that the side chain is connected to the amide nitrogen rather than the a carbon atom. Peptoids are typically resistant to proteases and other modifying enzymes and can have a much higher cell permeability than peptides (see e.g. Kwon and Kodadek, J. Am. Chem. Soc. 2007; 129:1508-1509). A suitable antibody may in some embodiments also be a multispecific antibody that includes several immunoglobulin fragments.

[0090] An immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like functions may be PEGylated or hyperglycosylated if desired. In some embodiments a proteinaceous binding molecule with immunoglobulin-like functions is a fusion protein of one of the exemplary proteinaceous binding molecules above and an albumin-binding domain, for instance an albumin-binding domain of streptococcal protein G. In some embodiments, a proteinaceous binding molecule with immunoglobulin-like functions is a fusion protein of an immunoglobulin fragment, such as a single-chain diabody, and an immunoglobulin binding domain, for instance a bacterial immunoglobulin binding domain. As an illustrative example, a single-chain diabody may be fused to domain B of staphylococcal protein A as described by Unverdorben et al., Protein Engineering, Design & Selection 2012; 25:81-88.

[0091] An immunoglobulin may be monoclonal or polyclonal. The term“polyclonal” refers to immunoglobulins that are heterogenous populations of immunoglobulin molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal immunoglobulins, one or more of various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species.“Monoclonal immunoglobulins", also called“monoclonal antibodies”, are substantially homogenous populations of immunoglobulins to a particular antigen. They may be obtained by any technique which provides for the production of immunoglobulin molecules by continuous cell lines in culture. Monoclonal immunoglobulins may be obtained by methods well known to those skilled in the art (see for example, Kohler et al., Nature (1975) 256, 495-497, and U.S. Patent No. 4,376,1 10). An immunoglobulin or immunoglobulin fragment with specific binding affinity only for e.g. CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138 can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art enable production of both immunoglobulins or immunoglobulin fragments and proteinaceous binding molecules with immunoglobulin-like functions, in both prokaryotic and eukaryotic organisms.

[0092] In more detail, an immunoglobulin may be isolated by comparing its binding affinity to a protein of interest, e.g. CD3, with its binding affinity to other polypeptides. Humanized forms of the antibodies of the present invention may be generated using one of the procedures known in the art such as chimerization or CDR grafting. In general, techniques for preparing monoclonal antibodies and hybridomas are well known in the art. Any animal such as a goat, a mouse or a rabbit that is known to produce antibodies can be immunized with the selected polypeptide, e.g. CD3. Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of polypeptide used for immunization and the immunization regimen will vary based on the animal which is immunized, including the species of mammal immunized, its immune status and the body weight of the mammal, as well as the antigenicity of the polypeptide and the site of injection.

[0093] As indicated above, a detectable marker may be coupled to a binding partner of CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138, as the case may be, or a molecule that forms a complex with the binding partner of CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138. In some embodiments, a detectable marker being coupled to a binding partner may refer to a“fluorescently labelled binding partner”. A respective detectable marker, which may be coupled to a binding partner of CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138, or a molecule that forms a complex therewith, may be an optically detectable label, a fluorophore, or a chromophore. Examples of suitable labels include, but are not limited to, an organic molecule, an enzyme, a radioactive, fluorescent, and/or chromogenic moiety, a luminescent moiety, a hapten, digoxigenin, biotin, a metal complex, a metal and colloidal gold. Accordingly an excitable fluorescent dye, a radioactive amino acid, a fluorescent protein or an enzyme may for instance be used to detect e.g. the level of CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and/or CD138. Examples of suitable fluorescent dyes include, but are not limited to, Krome Orange (KrO), fluorescein (FITC), fluorescein isothiocyanate, 5,6- carboxymethyl fluorescein, Cascade Blue®, Oregon Green®, Texas red, nitrobenz-2-oxa-1 ,3- diazol-4-yl, coumarin, dansyl chloride, rhodamine, amino-methyl coumarin, DAPI, Eosin, Erythrosin, BODIPY®, pyrene, lissamine, xanthene, acridine, a fluorescent brightener (PB), an oxazine, phycoerythrin, a Cy dye such as Cy3, Cy3.5, Cy5, Cy5PE, Cy5.5, Cy7, Cy7PE or Cy7APC, an Alexa dye such as Alexa 647, Alexa 750 or Alexa 700, and NBD (Naphthol basic dye). Examples of suitable fluorescent protein include, but are not limited to, EGFP, emerald, EYFP, a phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC), Monomeric Red Fluorescent Protein (mRFP), mOrange, mPlum and mCherry. In some embodiments a reversibly photoswitchable fluorescent protein such as Dronpa, bsDronpa and Padron may be employed (Andresen, M., et al., Nature Biotechnology (2008) 26, 9, 1035). Regarding suitable enzymes, alkaline phosphatase, soybean peroxidase, or horseradish peroxidase may serve as a few illustrative examples. In a further embodiemt, tandem conjugates such as PE-Cy5.5 or PE-Cy7 may also be used. In some embodiments a method of detection may include electrophoresis, HPLC, flow cytometry, fluorescence correlation spectroscopy or a modified form of these techniques. Some or all of these steps may be part of an automated separation/detection system.

[0094] In a preferred example, KrO may be used as a detectable marker (fluorescent dye) of the binding partner for CD45. PE-Cy5.5 may be used as a detectable marker (fluorescent dye) of the binding partner for CD3. APC may be used as a detectable marker (fluorescent dye) of the binding partner for CD4. PB may be used as a detectable marker (fluorescent dye) of the binding partner for CD8. FITC may be used as a detectable marker (fluorescent dye) of the binding partner for CD14. AF750 may be used as a detectable marker (fluorescent dye) of the binding partner for CD16. AF700 may be used as a detectable marker (fluorescent dye) of the binding partner for CD19. PE-Cy7 may be used as a detectable marker (fluorescent dye) of the binding partner for CD56. PE may be used as a detectable marker (fluorescent dye) of the binding partner for CD138.

[0095] The inventors surprisingly found out that only the combination of said defined cell ratios according to the present invention is indicative for whether or not said subject according to the present invention suffers from MS. The inventors also determined the ratio of activated T cells to non-activated T cells. This was achieved by detecting the level of activated and non-activated T cells using flow cytometry, preferably FACS. Detection of the level of activated and non- activated T cells was performed by measuring HLA-DR on CD45+ T cells, preferably using a binding partner for HLA-DR. HLA-DR is an MHC class II cell surface receptor. Only a limited number of cell types express MHC class II molecules; professional APCs, such as B cells, macrophages, Langerhans cells, and dendritic cells, display constitutive expression of MHC class II, while non-professional APCs, such as fibroblasts and T cells, acquire MHC class II expression under certain circumstances. Activated T cells may be identified by detecting HLA- DR on their cell surface, whereas non-activated T cells do not express HLA-DR on their surface. The inventors clearly found that this specific ratio of activated to non-activated T cells is not of importance for the indication of whether or not said subject suffers from MS (see also Table 1 and Fig. 10).

[0096] Further, according to the present invention, the method of the invention further comprises determining whether or not an immunoglobulin index (Ig index, preferably a IgG index) is elevated and/or whether oligoclonal bands (OCB, also known as oligoclonal IgG bands) may be detected in said test CSF sample. Commercially available clinical assay kits known to the person skilled in the art may be used to perform these analyses. In MS, CSF exhibits several disease-associated changes such as an increased CSF Ig index and OCB in the CSF (Kivisakk et al. 2003, Proc. Natl. Acad. Sci. U. S. A. 100, 8389-94).

[0097] OCB are bands of immunoglobulins, in particular oligoclonal immunoglobulins, that are seen when a patient's blood serum or cerebrospinal fluid (CSF) may be analyzed. Two methods of analysis for detecting OCB are possible, but not limited to: (a) protein electrophoresis, a method of analyzing the composition of fluids, also known as "agarose gel electrophoresis/Coomassie Blue staining"; and (b) the combination of protein separation by isoelectric focusing followed by immunoblotting (such as silver staining). In a preferred embodiment, the second method (b) for detecting OCB is applied in the present invention. In MS, normally only OCBs made of immunoglobulin G antibodies are considered, though sometimes other proteins can be taken into account, like lipid-specific immunoglobulin M. The presence of these IgM OCBs is associated with a more severe course of MS (Ferraro et al. 2015, Neuroimmunology. 283: 64-69). Because of the high sensitivity of CSF OCB in MS as well as its high specificity in the appropriate clinical setting, examination of CSF for OCB of IgG class may be highly recommended to support the diagnosis of MS including identify patients with clinically isolated syndrome (CIS) at increased risk of developing MS. In general, more than 95% of patients with multiple sclerosis (MS) have CSF OCB of IgG class, thereby providing powerful evidence for the diagnosis of MS. In fact, such immunoglobulins are thought to be produced locally by clonally expanded B cells in the CSF or meninges in MS and may recognize ubiquitous self-proteins. Thus, an importance of B cells in MS may be suggested by the presence of oligoclonal immunoglobulins (OCB).

[0098] Normally, proteins should not accumulate in the CSF. However, proteins such as Igs passaging from the blood into the CSF may be due to barrier disturbances of the blood- brain-barrier. The more permeable the blood-brain-barrier is, the more proteins (e.g., albumin) may be detected in the CSF. Another reason for detecting proteins such as Igs in the CSF is due to pathological processes of certain diseases including MS. However, if Igs are increased in said CSF, albumin concentration may also be detected to observe whether Igs may passage from the blood due to barrier disturbances of the blood-brain barrier or whether Igs may be produced in the CSF, which would correlate to a pathological process of certain diseases including, but not limited to, MS.

[0099] Therefore, the Ig index being additionally detected in the method of the present invention may be determined by comparing the Ig-ratio (in relation) to the albumin-ratio. As it may be done for determining the albumin ratio, the Ig-ratio may be determined by measuring the concentration / amount of Igs in the CSF and the concentration / amount of Igs in the blood serum. Then, the Ig concentration / amount of CSF is divided by the Ig concentration / amount of the blood serum, thereby obtaining the Ig-ratio. In some embodiments, the IgG ratio is of most importance in the method of the present invention. Thus, in a preferred embodiment, the concentration / amount of IgGs in the CSF and the concentration / amount of IgGs in the blood serum is measured. Then, the IgG concentration / amount of CSF is divided by the IgG concentration / amount of the blood serum, thereby obtaining the IgG-ratio. Elevation of IgG levels in the CSF of patients with MS is due to local central nervous system (CNS) synthesis of IgG. It may also be elevated in other inflammatory diseases such as neurosyphilis, acute inflammatory polyradiculoneuropathy, subacute sclerosing panencephalitis. By dividing the Ig-ratio (in particular the IgG-ratio) by the albumin-ratio, the Ig index (in particular the IgG index) may be obtained.

[00100] Due to lower diagnostic sensitivity, IgG index cannot replace the detection of CSF OCB in the diagnosis of MS. However, if said index is elevated in CSF it may be used as an additional evidence for an augmented B-cell response that is compatible with MS. CSF IgG index is positive (elevated) in approximately 80% of patients with MS.

[00101] The term“elevated immunoglobulin index (Ig index) - in particular IgG index-” as used herein and throughout the entire description refers to an Ig (or IgG) index value, which is above a certain reference value. In a preferred embodiment, said reference value is from about 0.12 to about 1.5, from about 0.15 to about 1.2, preferably from about 0.2 to about 0.9, preferably from about 0.25 to about 0.72, more preferably from about 0.3 to about 0.6, most preferably about 0.12, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, about 1 , about 1.1 , about 1.2, or about 1.5. An Ig index (in particular an IgG index) may also be elevated in the context of the present invention, if said Ig index (in particular of IgG index) deviates from a certain reference value. In another embodiment, said Ig index (in particular of IgG index) deviates from a certain reference value by about 0.1 %, about 0.2% about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. The greater the deviation of said Ig index (in particular said IgG index) from said reference value, the greater the possibility that an Ig index (in particular an IgG index) is elevated.

[00102] The respective method according to the present invention may also involve analysis of one or more samples from the subject in vitro. Typically the sample is, essentially consists of, or includes CSF from the subject. The term“essentially consists of” is understood to allow the presence of additional components in said CSF sample or a composition that do not affect the properties of the sample or a composition. Examples of additional components may include, but are not limited to certain types of media, or any type of buffer.

[00103] The sample used in the method of the present invention is a CSF sample. The CNS is enveloped and protected by CSF that provides a unique diagnostic option in clinical neurology. CSF is a clear, colorless body fluid found in the brain and spinal cord. It acts as a buffer for the brain, providing basic mechanical and immunological protection to the brain and serving a vital function in cerebral autoregulation of cerebral blood flow. CSF is derived from blood plasma and is largely similar to it. However, CSF is nearly protein-free compared with plasma and has some different electrolyte levels. In general, CSF is substantially free of red blood cells (erythrocytes), and at most contains a few white blood cells (leukocytes). In the present invention said CSF sample may be disclosed as test CSF sample meaning that said sample obtained from a subject according to the present invention is analyzed by determining said specific cell ratios as defined elsewhere herein, wherein the combination of said defined ratios is indicative whether or not said subject may suffer from MS.

[00104] The methods of the invention may include providing a sample from the subject or obtaining said sample from the subject. The sample may be obtained by lumbar puncture. Lumbar puncture may be performed under sterile conditions by inserting a needle into the subarachnoid space, preferably between the third and fourth lumbar vertebrae. Then, CSF may be extracted through the needle, and tested. In a preferred embodiment a 20G Sprotte Canulae (Pajunk Medical) may be used for performing a lumbar puncture.

[00105] The sample may have been taken at any desired point in time before carrying out the method of the invention. Generally a time interval between taking the sample and carrying out the method of the invention is selected to allow analysis of viable cells. Cell viability can readily be determined using standard methods known in the art, e.g. neutral red uptake (NRU) or water soluble tetrazolium (WST-1 ). It is within the skilled artisan’s experience to determine a respective time interval during which T cells in a sample can be expected to remain viable.

[00106] It is envisaged by the invention that the sample may have been taken on the same or on the previous day, such as about 48 hours, about 42 hours, about 36 hours, about 30 hours, about 28 hours, about 24 hours, about 18 hours, about 15 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours or less before the methods of the invention are being carried out. Also envisaged by the invention is that the sample may have been taken within a period of up to about 48 hours, i.e. 0 to about 48 hours, to about 42 hours, to about 36 hours, to about 30 hours, to about 28 hours, to about 24 hours, to about 18 hours, to about 15 hours or 0 to about 12 hours before the methods of the invention are being carried out. The subject, also addressed as a patient or an individual herein, from which/whom the sample has been obtained is an animal, generally a mammal.

[00107] The invention also contemplates that the sample from the individual may be a frozen sample. Generally the sample may be frozen within the above detailed time intervals, e.g. 0 to about 48 or 0 to about 42 hours, and/or at the above exemplified time points, such as about 48 hours, about 36 hours or less, after the sample has been obtained from the individual. A frozen sample may be formed by freezing an obtained sample after adding a cryoprotective agent such as DMSO, glycerol and/or hydroxyethyl starch. As an illustrative example DMSO may be used in a final concentration in the range from about 2% to about 10 %, such as about 2%, about 4%, about 5% or about 10% DMSO. Typically the sample is then frozen at a controlled rate to a temperature less than -50°C, whereafter the sample may for instance be stored, including long-term storage, at a temperature below -130°C such as -160°C, e.g. in liquid nitrogen for extended periods of time.

[00108] According to the present invention, the method of the invention of diagnosing the subject of the invention with MS further comprises the steps of multiplying

i) the ratio of the level of NK cells to the level of CD4+ T cells with an adjustment factor of preferably about 18 to about 74;

ii) the ratio of the level of B cells to the level of CD4+ T cells with an adjustment factor of preferably about 25 to about 101 ;

iii) the ratio of the level of CD8+ T cells to the level of CD4+ T cells with an adjustment factor of preferably about 2 to about 8;

iv) the ratio of the level of CD14+CD16- monocytes to the level of CD14+CD16+ monocytes with an adjustment factor of preferably about 0.6 to about 2.3. [00109] After the specific cell ratios as defined elsewhere herein have been determined in said test CSF sample being obtained from said subject, each cell ratio may be multiplied with an adjustment factor. This step refers to a normalization of each ratio. The application of said normalizing step is of great importance for the method of diagnosing a subject with MS, since the application of the absolute values of each defined ratios are not highly reliable on and should not be used.

[00110] The term“adjustment factor” as used herein and through the entire description refers to the reciprocal of each group average value of each ratio, which may be defined by the method of determining an adjustment factor as described below.

[00111] Said adjustment factor being multiplied with the ratio of the level of NK cells to the level of CD4+ T cells may be at least about 18, at least about 21 , at least about 25, at least about 29, at least about 32, at least about 34, at least about 35; or be any number in the range of about 18 to about 74, preferably about 21 to about 65, preferably about 25 to about 55, preferably about 29 to about 46, preferably about 32 to about 42, preferably about 34 to about 41 , preferably about 35 to about 39, or most preferably about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 35.5, about 36, about 36.5, about 36.6, about 36.7, about 36.8, about 36.85, about 36.86, about 36.9, about 37, about 37.5, about 38, about 38.5, about 39, about 40, about 41 , about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51 , about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61 , about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71 , about 72, about 73, about 74. In a preferred embodiment, said adjustment factor being multiplied with the ratio of the level of NK cells to the level of CD4+ T cells may be about 36.86.

[00112] Said adjustment factor being multiplied with the ratio of the level of B cells to the level of CD4+ T cells may be at least about 25, at least about 29, at least about 34, at least about 40, at least about 44, at least about 46, at least about 48; or be any number in the range of about 25 to about 101 , preferably about 29 to about 88, preferably about 34 to about 75, preferably about 40 to about 63, preferably about 44 to about 58, preferably about 46 to about 55, preferably about 48 to about 53, or most preferably about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 31 , about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 , about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 48, about 49, about 49.5, about 50, about 50.1 , about 50.2, about 50.3, about 50.32, about 50.35, about 50.5, about 51 , about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61 , about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71 , about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81 , about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91 , about 92, about 93, about 94, about 95, about 95, about 96, about 97, about 98, about 99, about 100, about 101. In a preferred embodiment, said adjustment factor being multiplied with the ratio of the level of B cells to the level of CD4+ T cells may be about 50.32.

[00113] Said adjustment factor being multiplied with the ratio of the level of CD8+ T cells to the level of CD4+ T cells may be at least about 2, at least about 2.2, at least about 2.5, at least about 3.0, at least about 3.3, at least about 3.5, at least about 3.6; or be any number in the range of about 2 to about 8, preferably about 2.2 to about 6.7, preferably about 2.5 to about 5.7, preferably about 3.0 to about 4.8, preferably about 3.3 to about 4.4, preferably about 3.5 to about 4.2, preferably about 3.6 to about 4.0, or most preferably about 2, about 2.1 , about 2.2, about 2.3, about 2.4, about 2.5, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1 , about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.81 , about 3.82, about 3.83, about 3.84, about 3.85, about 3.9, about 4.0, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0.

In a preferred embodiment, said adjustment factor being multiplied with the ratio of the level of CD8+ T cells to the level of CD4+ T cells may be about 3.81.

[00114] Said adjustment factor being multiplied with the ratio of the level of CD14+CD16- monocytes to the level of CD14+CD16+ monocytes may be at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1.0, at least about 1.1 ; or be any number in the range of about 0.6 to about 2.3, preferably about 0.7 to about 2.0, preferably about 0.8 to about 1.7, preferably about 0.9 to about 1.5, preferably about 1.0 to about 1.3, preferably about 1.1 to about 1.3, preferably about 1.1 to about 1.2, or most preferably about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1 , about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1 , about 2.2, about 2.3. In a preferred embodiment, said adjustment factor being multiplied with the ratio of the level of CD14+CD16- monocytes to the level of CD14+CD16+ monocytes may be about 1.16.

[00115] Additionally, the present invention further comprises a method of determining an adjustment factor as defined above for the method of the invention. [00116] First, the method may comprise the steps of

a) determining

i) the ratio of the (detected) level of NK cells to the (detected) level of CD4⁺ T cells; ii) the ratio of the (detected) level of B cells to the (detected) level of CD4⁺ T cells; iii) the ratio of the (detected) level of CD8⁺ cells to the (detected) level of CD4⁺ T cells, and/or

iv) the ratio of the (detected) level of CD14⁺CD16 monocytes to the (detected) level of CD14⁺CD16⁺ monocytes;

in a reference group of cerebrospinal fluid (CSF) samples comprising at least one sample obtained from a subject having multiple sclerosis and at least one sample obtained from a healthy subject.

[00117] Said reference group as used herein and throughout the entire description consisting of CSF samples only comprises at least one, such as one, two, three, four five, six, seven, eight, nine, ten, fifteen, twenty, twenty one, twenty two, twenty three, twenty four, twenty five, thirty, fourty, fifty, sixty, seventy, or more sample(s) being obtained / being provided from a subject having multiple sclerosis and at least one, such as one, two, three, four five, six, seven, eight, nine, ten, fifteen, twenty, twenty one, twenty two, twenty three, twenty four, twenty five, thirty, fourty, fifty, sixty, seventy, or more sample(s) being obtained / being provided from a healthy subject. Thus, the ratio defined in a) i), the ratio defined in a) ii), the ratio defined in a) iii), and the ratio defined in a) iv) may be determined for the at least one sample being obtained from a subject having MS and may be determined for the at least one sample obtained from the healthy subject. Also comprised herein, is that the ratio defined in a) i), the ratio defined in a) ii), the ratio defined in a) iii), or the ratio defined in a) iv) may be determined for the at least one sample being obtained from a subject having MS and may be determined for the at least one sample obtained from the healthy subject. The“at least one sample obtained from a subject having multiple sclerosis” refers to test sample. The “at least one sample obtained from a healthy subject” refers to a control sample. As an illustrative example the reference group may comprise three CSF control samples and three CSF test samples. For each CSF control sample each ratios defined in a) i) to a) iv) mentioned above may be determined, thereby determining six different ratio values for each defined ratio.

[00118] Second, the method of determining said adjustment factor as defined for the method of the invention may comprise calculating a group average for any one or all of the ratios of a) i) to a) iv) being determined in said reference group of CSF samples. According to the invention, a group average is calculated by summing up each ratio value for either any one or all of the defined ratios and then dividing said summed up ratio values of any one or all of the defined ratios by the number / amount of CSF samples being examined / determined in said reference group. The value being calculated thereby refers to the group average value for any one or all of the ratios being determined / examined in said reference group of CSF samples.

[00119] According to the illustrative example above, the group average may be calculated by summing up the six ratio values for any one of the ratios defined in a) i) to a) iv) and then dividing any one of the summed up ratio values for any one of the defined ratios by the number of six (six samples which have been examined comprising three control and three test samples). According to the illustrative example above, the group average may be calculated by summing up six ratio values for all of the ratios defined in a) i) to a) iv) and then dividing each summed up ratio value for all of the defined ratios by the number of six (six samples which have been examined comprising three control and three test samples).

[00120] Third, the method of determining said adjustment factor as defined for the method of the invention may comprise calculating the reciprocal value of each group average value being calculated above, thereby obtaining said adjustment factor.

[00121] According to the invention, the reciprocal value may be expressed as the quotient of the value of 1 divided by each group average value for any one or all of the ratios as defined above. According to the illustrative example above, each reciprocal value of any one of the defined ratio is calculated by dividing the value of 1 by the group average value for any one of the defined ratios. According to the illustrative example above, each reciprocal value for all of the defined ratios is calculated by dividing the value of 1 by the group average value for all of the defined ratios, thereby obtaining four different reciprocal values.

[00122] According to the invention, the ratio of the number of samples obtained from a subject having multiple sclerosis to the number of samples obtained from a healthy subject being comprised in the reference group may be between about 30% to about 70%, preferably about 35% to about 65%, preferably about 40% to about 60%, preferably about 45% to about 55%, and about 70% to about 30%. The ratio of the number of samples obtained from a subject having multiple sclerosis to the number of samples obtained from a healthy subject being comprised in the reference group may be between about 30% to about 70% and about 70% to about 30%, preferably about 65% to about 35%, preferably about 60% to about 40%, preferably about 55% to about 45%.

[00123] As used herein, for the method of determining said adjustment factor, a healthy subject may be a subject not suspected to suffer / is not suffering from MS. Preferably, the healthy subject has idiopathic intracranial hypertension (IIH). IIH is a condition characterized by increased intracranial pressure (pressure around the brain) without a detectable cause. Main symptoms are headache, vision problems and shoulder pain.

[00124] According to the present invention, the method of the invention may also comprise the steps of multiplying

i) the ratio of the level of NK cells to the level of CD4+ T cells with a weighting factor;

ii) the ratio of the level of B cells to the level of CD4+ T cells with an a weighting factor; iii) the ratio of the level of CD8+ T cells to the level of CD4+ T cells with a weighting factor; iv) the ratio of the level of CD14+CD16- monocytes to the level of CD14+CD16+ monocytes with a weighting factor.

[00125] The term “weighting factor” refers to a value being calculated by multiplying each reciprocal value of each group average value of each defined ratio as defined above with a weighting score of each ratio. The method of the invention also comprises that there might be a different weighting between each defined ratio. As an illustrative example, the ratio of the level of CD8+ T cells to the level of CD4+ T cells may have compared to the other ratios as defined a greater weighting. Thus, said specific ratio would get assigned a different (greater) weighting score (e.g., a weighting score value of 2) compared to the other defined ratios (e.g. level of NK to the level of CD4+ T cells), which would get assigned a lower weighting score (e.g., a weighting score value of 1 ). According to the illustrative example, the weighting factor for the ratio of the level of CD8+ T cells to the level of CD4+ T cells would be calculated by multiplying the weighting score value of 2 with the already calculated reciprocal value of the group average for this specific ratio. In some illustrative examples, the method of the invention does not include multiplying the ratio of the level of NK cells to the level of CD4+ T cells, the ratio of the level of B cells to the level of CD4+ T cells, the ratio of the level of CD8+ T cells to the level of CD4+ T cells, and/or the ratio of the level of CD14+CD16- monocytes to the level of CD14+CD16+ monocytes with a weighting factor.

[00126] After having multiplied each ratio as defined elsewhere herein with an adjustment factor or with a weighting factor being determined above, the method of the present invention may further comprise the steps of summing up each adjusted (or weighted) ratio, thereby obtaining a basic composite score. If said defined ratios may be multiplied with an adjustment factor as defined elsewhere herein, each ratio may refer to an adjusted ratio. If said defined ratios may be multiplied with a weighting factor as defined elsewhere herein, each ratio may refer to a weighted ratio.

[00127] The present inventors found out that by generating the basic composite score as defined above using a multiplication of each adjusted (or weighted) ratio, the“multiplied” basic composite score was indeed worse for diagnosing a subject with MS (see Fig. 10 and Table 1), since outliers of highly fluctuating values gain more importance by using multiplication.

Therefore, it has been demonstrated by the inventors that summing up each adjusted (or weighted) ratio generates a more reliable basic composite score for diagnosing a subject with

MS.

Table 1; Overview of ROC analysis with different composite scores.

[00128] Optionally, the method of the present invention may further comprise the step of adding a value of 1 to the basic composite score as defined above in each case if an elevated immunoglobulin index (Ig index, in particular IgG index) or oligoclonal bands have been detected in said test CSF sample, thereby obtaining an extended composite score.

[00129] The term“composite score” refers to a score which is based on more than one, such as four, specific, but different cell ratios defined elsewhere herein and which is calculated by the method of the invention. There are two different composite scores, one being called“basic composite score” and the other being called “extended composite score”. The difference between these composite scores only lies in the optional detection step of an elevated Ig index (in particular IgG index) and/or oligoclonal bands for the extended composite score (see Fig. 11 and 12).

[00130] If for said test CSF sample being examined no Ig index, in particular no IgG index, may be elevated as defined elsewhere and no oligoclonal bands (oligoclonal immunoglobulins) (OCM) may have been detected as defined elsewhere herein, a value of 0 is added to the basic composite score, thus obtaining an extended composite score which is identical to said basic composite score. If for said test CSF sample being examined an Ig index, in particular an IgG index, may be elevated and detected as defined elsewhere herein, a value of 1 is added to the basic composite score, thus obtaining an extended composite score. If for said test CSF sample being examined oligoclonal bands (oligoclonal immunoglobulins; OCM) may have been detected as defined elsewhere herein, a value of 1 is added to the basic composite score, also obtaining an extended composite score. If for said test CSF sample being examined an Ig index, in particular an IgG index, may be elevated as defined elsewhere and oligoclonal bands (oligoclonal immunoglobulins) (OCM) may have been detected as defined elsewhere herein, a value of 2 is added to the basic composite score, again obtaining an extended composite score. By adding either a value of 0, a value of 1 or a value of 2, an extended composite score may be obtained / generated / produced, which has also considered further diagnostic analyses (e.g, Ig index and/or OCM) for diagnosing a subject with MS.

[00131] As an illustrative example, an example calculation may be demonstrated below for determining a composite score for the method of diagnosing a subject with MS.

First, the cell levels may be detected in a test CSF sample obtained from a subject using flow cytometry:

%CD4 60.1 % %CD4 60.1 % %CD4 60.1 % %CD16+ 12.6%

%NK 1.4% %B 0.25% %CD8 9.41 % %CD16- 3.1 %

Then, each ratio as defined elsewhere herein may be determined / calculated by comparing each specific cell level to one another, meaning dividing the level of NK cells by the level of CD4+ T cells, the level of B cells by the level of CD4+ T cells, the level of CD8+ T cells by the level of CD4+ T cells, and the level of classical monocytes by the level of non-classical monocytes. ratio: 0.0233 0.00428 0.156 0.245

Next, each ratio value defined above may be multiplied with an adjustment factor. Preferably, for said ratio of NK:CD4 an adjustment factor of 36.86 may be used. Preferably, for said ratio of B:CD4 an adjustment factor of 50.32 may be used. Preferably, for said ratio of CD8:CD4 an adjustment factor of 3.81 may be used. Preferably, for said ratio of CD16-:CD16+ an adjustment factor of 1.16 may be used.

According to the abovementioned ratios, each adjusted ratios may be calculated:

0.858 0.215 0.594 0.284

By adding those adjusted ratios, a basic composite score of 1.951 may be obtained. As an optional step, said sample may be examined for the detection of an elevated immunoglobulin index (Ig index, in particular IgG index) or oligoclonal bands, which may be negative for said particular CSF sample, thereby obtaining an extended composite score of 1.951 , which may be identical to said basic composite score.

[00132] Further, the method of the present invention may additionally comprise the further step of comparing said basic composite score or said extended composite score determined in said test CSF sample with a reference value.

[00133] The term “reference value” is understood to generally refer to a qualitative or quantitative value. Thus, if the reference value is understood to refer to a qualitative value, said basic composite score or said extended composite score may be above said reference value. Also comprised herein is, if the reference value is understood to refer to a quantitative value, said basic composite score or said extended composite score may deviate from said reference value. In another embodiment, if the reference value is understood to refer to a quantitative value, said basic composite score or said extended composite score may deviate from said reference value by about 0.1 %, about 0.2% about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. The greater the deviation of said basic composite score or said extended composite score from said reference value, the higher the possibility that a subject of the present invention may suffer from MS or that said subject may be diagnosed with MS.

[00134] In comparison, a threshold value is understood to generally refer to a qualitative value. If said basic composite score or said extended composite score may be above said specific value, being compared to, the term“threshold value” may be used interchangeably with the term“reference value”. The term“a reference” may be used interchangeably with the term“a reference value". The term “a threshold” may be used interchangeably with the term “a threshold value”.

[00135] Selecting a diagnostic reference / threshold involves, among other things, consideration of the probability of disease, distribution of true and false diagnoses at different test references / thresholds, and estimates of the consequences of treatment (or a failure to treat) based on the diagnosis. Suitable references / thresholds may be determined in a variety of ways. [00136] For example population studies may be used to select a reference / threshold value. Receiver Operating Characteristic ("ROC") is often used to select a reference / threshold able to best distinguish a well responding subpopulation (a control sample not being diagnosed with MS) from a poorly responding subpopulation (a test CSF sample being diagnosed with MS). A false positive in this case occurs when a person tests positive (poor responder or diagnosed with MS), but actually is a good responder and thus does not suffer from MS. A false negative, on the other hand, occurs when the person tests negative (good responder or not being diagnosed with MS), when it actually suffers from MS. To draw a ROC curve, the true positive rate (TPR) and false positive rate (FPR) are determined as the decision reference / threshold is varied continuously. Since TPR is equivalent with sensitivity and FPR is equal to 1 - specificity, the ROC graph is sometimes called the sensitivity vs (1 - specificity) plot. A perfect test will have an area under the ROC curve (AUC) of 1.0; a random test will have an area of 0.5. Preferably, the tests described herein provide a ROC curve area greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95. A reference / threshold value is selected to provide an acceptable level of specificity and sensitivity. A reference / threshold that can provide an acceptable level of specificity and sensitivity in separating a population of subjects into“bins” such as a“first” subpopulation (e.g., which is diagnosed with MS) and a“second” subpopulation which is not diagnosed with MS. A reference / threshold value is selected to separate this first and second population by one or more of the following measures of test accuracy: an odds ratio greater than 1 , preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a specificity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, still more preferably at least about 0.8, even more preferably at least about 0.9 and most preferably at least about 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, yet more preferably greater than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9, and most preferably greater than about 0.95;

a sensitivity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, still more preferably at least about 0.8, even more preferably at least about 0.9 and most preferably at least about 0.95, with a corresponding specificity greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, yet more preferably greater than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9, and most preferably greater than about 0.95;

at least about 75% sensitivity, combined with at least about 75% specificity; a positive likelihood ratio (calculated as sensitivity/(1 -specificity)) of greater than 1 , at least about 2, more preferably at least about 3, still more preferably at least about 5, and most preferably at least about 10; or a negative likelihood ratio (calculated as (1 -sensitivity )/specificity) of less than 1 , less than or equal to about 0.5, more preferably less than or equal to about 0.3, and most preferably less than or equal to about 0.1.

[00137] According to the present invention and in particular according to Table 1 , diagnosing a subject with MS by determining the extended composite score based on the ratios of NK:CD4+, B:CD4+, CD8+:CD4+ and of classicahnon-classical monocytes, also determining an elevated IgG index and oligoclonal bands may demonstrate a very sensitiv and specific test (e.g. AUC: 0.9176) in comparison to the corresponding basic composite score not determining an elevated IgG index and oligoclonal bands (e.g. AUC: 0.8618). With respect to diagnosing a subject with MS by determining the basic composite score based on the ratios of NK:CD4+, B:CD4+, CD8+:CD4+, activated :non-activated T cell, and of classicahnon-classical monocytes demonstrates a rather poor test depending on the fact that the ratio of activated to non-activated T cells has been considered (e.g. AUC: 0.8588). The most random test may be achieved by the diagnosis based on the determination of the basic composite score based on the two ratios of B:CD4+ and of classical:non-classical monocytes (AUC: 0.8324) (see also Fig. 11 and 12 for supporting these particular findings of the present invention).

[00138] In addition to reference / threshold comparisons, other methods for correlating assay results to a patient selection or classification (e.g. likelihood of being a good responder or a poor responder) include decision trees, rule sets, Bayesian methods, and neural network methods. These methods can produce probability values representing the degree to which a subject belongs to one classification out of a plurality of classifications.

[00139] Measures of test accuracy may be obtained as described in Fischer et at., Intensive Care Med. 29: 1043-51 , 2003, and used to determine the effectiveness of a given biomarker. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. The area under the curve (“AUC”) of a ROC plot is equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. The area under the ROC curve may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.

[00140] As discussed above, suitable tests may exhibit one or more of the following results on these various measures: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding specificity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; at least 75% sensitivity, combined with at least 75% specificity; a ROC curve area of greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; an odds ratio different from 1 , preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a positive likelihood ratio (calculated as sensitivity/(1 - specificity)) of greater than 1 , at least 2, more preferably at least 3, still more preferably at least 5, and most preferably at least 10; and or a negative likelihood ratio (calculated as (1 - sensitivity)/specificity) of less than 1 , less than or equal to 0.5, more preferably less than or equal to 0.3, and most preferably less than or equal to 0.1.

[00141] The comparison to a reference / threshold value may be carried out manually, semi- automatically or in a fully automated manner. In some embodiments, the comparison may be computer assisted. A computer assisted comparison may employ values stored in a database as a reference for comparing an obtained value or a determined amount, for example via a computer implemented algorithm. Likewise, the comparison to a reference measurement may be carried out manually, semi-automatically or in a fully automated manner, including in a computer assisted manner. [00142] According to the present invention, if said basic composite score or said extended composite score may be above the reference value, it may be indicative for whether or not said subject suffers from MS. The present invention also comprises that if said basic composite score or said extended composite score may deviate from a reference value, it may be indicative for whether or not said subject may suffer from MS. In a preferred embodiment, if said basic composite score or said extended composite score may deviate from a reference value by about 0.1%, about 0.2% about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, or by about 100%, it may be indicative for whether or not said subject may suffer from MS. The greater the deviation of said basic composite score or said extended composite score from said reference value, the higher the possibility that the subject may be diagnosed with MS or that the subject may suffer from MS.

[00143] In a preferred embodiment, the reference value may be at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0, at least about 3.1 , at least about 3.2, at least about 3.3, at least about 3.4, at least about 3.5, at least about 3.6, at least about 3.7, at least about 3.8, at least about 3.9, at least about 4.0, at least about 4.1 , at least about 4.2, at least about 4.3, at least about 4.4, at least about 4.5, at least about 4.6, at least about 4.7, at least about 4.8, at least about 4.9, at least about 5.0, at least about 5.1 , at least about 5.2, at least about 5.3, at least about 5.4, at least about 5.5, at least about 5.6, at least about 5.7, at least about 5.8, at least about 5.9, at least about 6.0, or be any number in the range of about 2.5 to about 6.0, from about 2.5 to about 5.9, from about 2.5 to about 5.8, from about 2.5 to about 5.7, from about 2.5 to about 5.6, from about 2.5 to about 5.5, from about 2.5 to about 5.4, from about 2.5 to about 5.3, from about 2.5 to about 5.2, from about

2.5 to about 5.1 , from about 2.5 to about 5.0, from about 2.5 to about 4.9, from about 2.5 to about 4.8, from about 2.5 to about 4.7, from about 2.5 to about 4.6, from about 2.5 to about 4.5, from about 2.5 to about 4.4, from about 2.5 to about 4.3, from about 2.5 to about 4.2, from about

2.5 to about 4.1 , from about 2.5 to about 4.0, from about 2.5 to about 3.9, from about 2.5 to about 3.8, from about 2.5 to about 3.7, from about 2.5 to about 3.6, from about 2.5 to about 3.5, from about 2.5 to about 3.4, from about 2.5 to about 3.3, from about 2.5 to about 3.2, from about

2.5 to about 3.1 , from about 2.5 to about 3.0, or from about 2.6 to about 6.0, from about 2.6 to about 5.9, from about 2.6 to about 5.8, from about 2.6 to about 5.7, from about 2.6 to about 5.6, from about 2.6 to about 5.5, from about 2.6 to about 5.4, from about 2.6 to about 5.3, from about

2.6 to about 5.2, from about 2.6 to about 5.1 , from about 2.6 to about 5.0, from about 2.6 to about 4.9, from about 2.6 to about 4.8, from about 2.6 to about 4.7, from about 2.6 to about 4.6, from about 2.6 to about 4.5, from about 2.6 to about 4.4, from about 2.6 to about 4.3, from about

2.6 to about 4.2, from about 2.6 to about 4.1 , from about 2.6 to about 4.0, from about 2.6 to about 3.9, from about 2.6 to about 3.8, from about 2.6 to about 3.7, from about 2.6 to about 3.6, from about 2.6 to about 3.5, from about 2.6 to about 3.4, from about 2.6 to about 3.3, from about

2.6 to about 3.2, from about 2.6 to about 3.1 , from about 2.6 to about 3.0, or from about 2.7 to about 6.0, from about 2.7 to about 5.9, from about 2.7 to about 5.8, from about 2.7 to about 5.7, from about 2.7 to about 5.6, from about 2.7 to about 5.5, from about 2.7 to about 5.4, from about

2.7 to about 5.3, from about 2.7 to about 5.2, from about 2.7 to about 5.1 , from about 2.7 to about 5.0, from about 2.7 to about 4.9, from about 2.7 to about 4.8, from about 2.7 to about 4.7, from about 2.7 to about 4.6, from about 2.7 to about 4.5, from about 2.7 to about 4.4, from about

2.7 to about 4.3, from about 2.7 to about 4.2, from about 2.7 to about 4.1 , from about 2.7 to about 4.0, from about 2.7 to about 3.9, from about 2.7 to about 3.8, from about 2.7 to about 3.7, from about 2.7 to about 3.6, from about 2.7 to about 3.5, from about 2.7 to about 3.4, from about

2.7 to about 3.3, from about 2.7 to about 3.2, from about 2.7 to about 3.1 , from about 2.7 to about 3.0, or from about 2.8 to about 6.0, from about 2.8 to about 5.9, from about 2.8 to about

5.8, from about 2.8 to about 5.7, from about 2.8 to about 5.6, from about 2.8 to about 5.5, from about 2.8 to about 5.4, from about 2.8 to about 5.3, from about 2.8 to about 5.2, from about 2.8 to about 5.1 , from about 2.8 to about 5.0, from about 2.8 to about 4.9, from about 2.8 to about

4.8, from about 2.8 to about 4.7, from about 2.8 to about 4.6, from about 2.8 to about 4.5, from about 2.8 to about 4.4, from about 2.8 to about 4.3, from about 2.8 to about 4.2, from about 2.8 to about 4.1 , from about 2.8 to about 4.0, from about 2.8 to about 3.9, from about 2.8 to about

3.8, from about 2.8 to about 3.7, from about 2.8 to about 3.6, from about 2.8 to about 3.5, from about 2.8 to about 3.4, from about 2.8 to about 3.3, from about 2.8 to about 3.2, from about 2.8 to about 3.1 , from about 2.8 to about 3.0, or from about 2.9 to about 6.0, from about 2.9 to about

5.9, from about 2.9 to about 5.8, from about 2.9 to about 5.7, from about 2.9 to about 5.6, from about 2.9 to about 5.5, from about 2.9 to about 5.4, from about 2.9 to about 5.3, from about 2.9 to about 5.2, from about 2.9 to about 5.1 , from about 2.9 to about 5.0, from about 2.9 to about

4.9, from about 2.9 to about 4.8, from about 2.9 to about 4.7, from about 2.9 to about 4.6, from about 2.9 to about 4.5, from about 2.9 to about 4.4, from about 2.9 to about 4.3, from about 2.9 to about 4.2, from about 2.9 to about 4.1 , from about 2.9 to about 4.0, from about 2.9 to about

3.9, from about 2.9 to about 3.8, from about 2.9 to about 3.7, from about 2.9 to about 3.6, from about 2.9 to about 3.5, from about 2.9 to about 3.4, from about 2.9 to about 3.3, from about 2.9 to about 3.2, from about 2.9 to about 3.1 , from about 2.9 to about 3.0, preferably about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1 , about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, most preferably about 2.9. It is understood that ranges herein include all values in between such as natural numbers, rational numbers or real numbers. In an even more preferred embodiment, the reference value for the basic and the extended composite score may be (at least) about 2.9. Thus, if said basic composite score may be above the reference (or threshold) value of about 2.9, it may be indicative that the subject suffers from MS. Put it differently, if said basic composite score may be above the reference (or threshold) value of about 2.9, the subject may be diagnosed with MS. If said extended composite score may be above the reference (or threshold) value of about 2.9, it may be indicative that the subject suffers from MS. Put it differently, if said extended composite score may be above the reference (or threshold) value of about 2.9, the subject may be diagnosed with MS.

[00144] In this context, the term“above a certain reference (or threshold) value” means any number, preferably being rounded up to two digits behind the comma, which is above (not equal to) the indicated number of the reference (or threshold) value (e.g. for example about 2.5). As an illustrative example, if said number may be about 2.51 , preferably being rounded up to two digits behind the comma, said number may be above the reference value of about 2.5.

Computer-implemented features

[00145] The subject-matter of the defined method steps of the present invention may also fully be carried out by computer program instructions running on means which, in the context of the invention, provide generic data processing functions. Such means may, for example, be embedded in a personal computer, smartphone, printer. A computer-implemented invention may therefore be one which involves the use of a computer, computer network or other programmable apparatus, where one or more features are realised wholly or partly by means of a computer program.

[00146] Thus, the present invention comprises a data processing system comprising a processor configured to perform a method comprising the steps of

a) obtaining

i) the detected level of NK cells;

ii) the detected level of CD4⁺ T cells;

iii) the detected level of B cells;

iv) the detected level of CD8⁺ cells;

v) the detected level of CD14⁺CD16 monocytes; and

vi) the detected level of CD14⁺CD16⁺ monocytes;

from a test CSF sample obtained from a subject;

b) determining i) the ratio of the level of NK cells of step ai) to the level of CD4⁺ T cells of step aii); ii) the ratio of the level of B cells of step aiii) to the level of CD4⁺ T cells of step aii); iii) the ratio of the level of CD8⁺ cells of step aiv) to the level of CD4⁺ T cells of step aii); and

iv) the ratio of the level of CD14⁺CD16 monocytes of step av) to the level of CD14⁺CD16⁺ monocytes of step avi);

C) multiplying

i) the ratio of the level of NK cells to the level of CD4⁺ T cells with an adjustment factor of preferably about 18 to about 74,

ii) the ratio of the detected level of B cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 25 to about 101 ,

iii) the ratio of the detected level of CD8⁺ cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 2 to about 8

iv) the ratio of the detected level of CD14⁺CD16 monocytes to the detected level of CD14⁺CD16⁺ monocytes with an adjustment factor of preferably about 0.6 to about 2.3;

d) summing up each adjusted ratio of step c i) - c iv), thereby obtaining the basic composite score; optionally

e) adding a value of 1 to the basic composite score of step d) in each case if an elevated immunoglobulin index or oligoclonal bands has been detected in said test CSF sample, thereby obtaining an extended composite score.

Further, the present invention comprises the data processing system comprising a processor configured to perform the abovementioned method further comprising the steps of

a) comparing said basic composite score or said extended composite score determined in said test CSF sample with a reference value (or threshold value); and

b) indicating whether or not said subject suffers from multiple sclerosis.

[00147] According to the present invention, said specific cell levels as defined above may be detected in a test CSF sample obtained from a subject according to the present invention using flow cytometry, in particular using a flow cytometry device. The detected level of specific cells being expressed in terms a decimal value or a percentage as described elsewhere herein may be entered into said data processing system comprising a processor which is configured to perform the abovementioned steps. In some embodiments, the step of obtaining said detected cell ratios as defined above may be an optional step. As used herein, by using the term “obtaining / obtain the detected level of specific cells" in this context means that the data for the detected level (in percentage) of defined cells being detected in a flow cytometry device as defined elsewhere herein is entered into the data processing system by any way known to a person skilled in the art. The step of determining each specific cell ratio as defined herein refers to comparing each specific level of cells to other specific level of cells as described herein applying division. The step of multiplying each determined cell ratio as defined herein with an adjustment factor refers to a normalizing step of each determined cell ratio as described herein. Also comprised herein, is multiplying each determined cell ratio with a weighting factor as defined elsewhere herein. The step of adding a value of 1 to the basic composite score in each case if an elevated immunoglobulin index or oligoclonal bands has been detected in said test CSF sample as described elsewhere herein, thereby obtaining an extended composite score may refer to an optional step. Another optional step before adding a value of 1 to the basic composite score as described above is the step of obtaining information regarding an elevated immunoglobulin index or the presence or absence of oligoclonal bands.

[00148] For the step of comparing said basic composite score or said extended composite score with a reference value (or threshold value) as used herein, it is of importance whether said basic composite score or said extended composite score is above or below the reference value (or threshold value). If said basic composite score or said extended composite score may be above the reference value (or threshold value) being defined elsewhere herein, it may be indicative that said subject may suffers from MS. If said basic composite score or said extended composite score may be below the reference value (or threshold value) being defined elsewhere herein, it may be indicative that said subject may not suffer from MS. Also the deviation of said basic composite score or said extended composite score being defined elsewhere herein from the reference value may be considered for the comparing step as defined above. The greater the deviation of said basic composite score or said extended composite score from said reference value, the higher the possibility that the subject may be diagnosed with MS or that the subject may suffer from MS.

[00149] Also comprised by the present invention is a data processing system comprising a processor configured to perform a method comprising the steps of

a) comparing a basic composite score or an extended composite score determined in a test CSF sample obtained from a subject, wherein said scores are being calculated by i) multiplying

xi) the ratio of the detected level of NK cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 18 to about 74,

xii) the ratio of the detected level of B cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 25 to about 101 ,

xiii) the ratio of the detected level of CD8⁺ cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 2 to about 8 xiv) the ratio of the detected level of CD14⁺CD16 monocytes to the detected level of CD14⁺CD16⁺ monocytes with an adjustment factor of preferably about 0.6 to about 2.3;

ii) summing up each adjusted ratio, thereby obtaining the basic composite score; optionally

iii) adding a value of 1 to the basic composite score of step ii) in each case if an elevated immunoglobulin index or oligoclonal bands has been detected in said test CSF sample, thereby obtaining an extended composite score;

with a reference value (or threshold value);

b) indicating whether or not said subject suffers from multiple sclerosis.

The definitions made above for the other data processing system may also apply mutatis mutandis to this defined data processing system.

[00150] A more meaningful characterization of modern digital data processing systems is the functional classification as either computational or input/output (“I/O") oriented. As the classifying labels imply, computational oriented data processing systems are designed primarily for performing long, complicated calculations. I/O oriented data processing systems are designed to handle large quantities of digital data, thereby requiring extensive I/O operations. Both data processing systems may be comprised by the present invention. The data processing system of the present invention may also be utilized as a peripheral subsystem of a larger computational computer. The structural design of a data processing system may necessarily directly be related to the functional use to which the data processing system is put. A data processing system of the present invention may refer to a programmable or programmed device / apparatus, where one or more features are realised wholly or partly by means of a computer program. A data processing system may include, but are not limited to, a computer, smartphone, tablet, chip.

[00151] As used herein, the term“processor” may refer to one functional element being a physical unit of a data processing system, which is configured by a computer program as defined elsewhere herein to perform the specified steps mentioned above.

[00152] Also comprised by the present invention is a data processing system comprising a processor configured to perform a method of determining an adjustment factor comprising the steps of

a) obtaining

i) the detected level of NK cells;

ii) the detected level of CD4⁺ T cells;

iii) the detected level of B cells; iv) the detected level of CD8⁺ cells;

v) the detected level of CD14⁺CD16 monocytes; and

vi) the detected level of CD14⁺CD16⁺ monocytes;

from a reference group of CSF samples comprising at least one sample obtained from a subject having multiple sclerosis and at least one sample obtained from a healthy subject;

b) determining

i) the ratio of the level of NK cells of step ai) to the level of CD4⁺ T cells of step aii); ii) the ratio of the level of B cells of step aiii) to the level of CD4⁺ T cells of step aii); iii) the ratio of the level of CD8⁺ cells of step aiv) to the level of CD4⁺ T cells of step aii), and/or

iv) the ratio of the level of CD14⁺CD16 monocytes of step av) to the level of CD14⁺CD16⁺ monocytes of step avi);

in said reference group of cerebrospinal fluid (CSF) samples;

c) calculating a group average for any one or all of the defined ratios above being determined in said reference group of CSF samples;

d) calculating the reciprocal value of each group average value defined above, thereby obtaining the adjustment factor.

[00153] In some embodiments, the step of obtaining said detected cell ratios as defined above may be an optional step. As used herein, by using the term“obtaining / obtain the detected level of specific cells” in this context means that the data for the detected level (in percentage) of defined cells being detected in a flow cytometry device as defined elsewhere herein is entered into the data processing system by any way known to a person skilled in the art.

[00154] The present invention further comprises the interaction between the data processing steps and other technical means such as a flow cytometry device.

[00155] Thus, the present invention comprises a flow cytometry device capable of detecting i) the level of NK cells;

ii) the level of CD4⁺ T cells;

iii) the level of B cells;

iv) the level of CD8⁺ cells;

v) the level of CD14⁺CD16 monocytes; and

vi) the level of CD14⁺CD16⁺ monocytes;

in a test CSF sample obtained from a subject, comprising the abovementioned data processing systems. [00156] Also comprised herein is a flow cytometry device capable of detecting

i) the level of NK cells;

ii) the level of CD4⁺ T cells;

iii) the level of B cells;

iv) the level of CD8⁺ cells;

v) the level of CD14⁺CD16 monocytes; and

vi) the level of CD14⁺CD16⁺ monocytes;

in a reference group of CSF samples comprising at least one sample obtained from a subject having multiple sclerosis and at least one sample obtained from a healthy subject, comprising the abovementioned data processing systems defined in [00152]

[00157] As defined above, said specific cell levels may be detected by using a flow cytometry device, preferably using flow cytometry analysis being combined with immunofluorescence as described elsewhere herein. In a preferred embodiment said flow cytometry device as defined above may be a FACS. In some embodiments said FACS may be connected to a computer, tablet, smartphone or any other technical device / apparatus being used as a further means for performing said FACS analysis. In another embodiment said FACS may not be connected to a computer, tablet, smartphone or any other technical device / apparatus being used as a further means for performing said FACS analysis.

[00158] The present invention also envisages a computer program comprising instructions to cause the data processing system as defined above or the flow cytometry device as defined above to execute the steps of

a) obtaining

i) the detected level of NK cells;

ii) the detected level of CD4⁺ T cells;

iii) the detected level of B cells;

iv) the detected level of CD8⁺ cells;

v) the detected level of CD14⁺CD16 monocytes; and

vi) the detected level of CD14⁺CD16⁺ monocytes;

from a test CSF sample obtained from a subject;

b) determining

i) the ratio of the level of NK cells of step ai) to the level of CD4⁺ T cells of step aii); ii) the ratio of the level of B cells of step aiii) to the level of CD4⁺ T cells of step aii); iii) the ratio of the level of CD8⁺ cells of step aiv) to the level of CD4⁺ T cells of step aii); and

iv) the ratio of the level of CD14⁺CD16 monocytes of step av) to the level of CD14⁺CD16⁺ monocytes of step avi); c) multiplying

i) the ratio of the level of NK cells to the level of CD4⁺ T cells with an adjustment factor of preferably about 18 to about 74,

ii) the ratio of the detected level of B cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 25 to about 101 ,

iii) the ratio of the detected level of CD8⁺ cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 2 to about 8

iv) the ratio of the detected level of CD14⁺CD16 monocytes to the detected level of CD14⁺CD16⁺ monocytes with an adjustment factor of preferably about 0.6 to about 2.3;

d) summing up each adjusted ratio of step c i) - c iv), thereby obtaining the basic composite score; optionally

e) adding a value of 1 to the basic composite score of step d) in each case if an elevated immunoglobulin index or oligoclonal bands has been detected in said test CSF sample, thereby obtaining an extended composite score.

Further, also comprised by the present invention is a computer program comprising instructions to cause the data processing system as defined above or the flow cytometry device as defined above to further execute the steps of

a) comparing said basic composite score or said extended composite score determined in said test CSF sample with a reference value (or threshold value); and

b) indicating whether or not said subject suffers from multiple sclerosis.

[00159] Again, in some embodiments, the step of obtaining said detected cell ratios as defined above may be an optional step. In other embodiments, another optional step before adding a value of 1 to the basic composite score as described above is the step of obtaining information regarding an elevated immunoglobulin index or the presence or absence of oligoclonal bands.

[00160] Also comprised by the present invention is a computer program comprising instructions to cause the data processing system or the flow cytometry device to execute the steps of a) comparing a basic composite score or an extended composite score determined in a test CSF sample obtained from a subject, wherein said scores are being calculated by i) multiplying

xi) the ratio of the detected level of NK cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 18 to about 74,

xii) the ratio of the detected level of B cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 25 to about 101 , xiii) the ratio of the detected level of CD8⁺ cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 2 to about 8

xiv) the ratio of the detected level of CD14⁺CD16 monocytes to the detected level of CD14⁺CD16⁺ monocytes with an adjustment factor of preferably about 0.6 to about

2.3;

ii) summing up each adjusted ratio, thereby obtaining the basic composite score; optionally

iii) adding a value of 1 to the basic composite score of defined above in each case if an elevated immunoglobulin index or oligoclonal bands has been detected in said test CSF sample, thereby obtaining an extended composite score;

with a reference value (or threshold value);

b) indicating whether or not said subject suffers from multiple sclerosis.

[00161] Everything being defined above for said data processing system ([00146]-[00151 ]) may apply mutatis mutandis to the corresponding computer program as defined above.

[00162] The present invention also envisages a computer program comprising instructions to cause the data processing system as defined in [00152] or the flow cytometry device as defined in [00156] above to execute the steps of

a) obtaining

i) the detected level of NK cells;

ii) the detected level of CD4⁺ T cells;

iii) the detected level of B cells;

iv) the detected level of CD8⁺ cells;

v) the detected level of CD14⁺CD16 monocytes; and

vi) the detected level of CD14⁺CD16⁺ monocytes;

from a reference group of CSF samples comprising at least one sample obtained from a subject having multiple sclerosis and at least one sample obtained from a healthy suject; b) determining

i) the ratio of the level of NK cells of step ai) to the level of CD4⁺ T cells of step aii); ii) the ratio of the level of B cells of step aiii) to the level of CD4⁺ T cells of step aii); iii) the ratio of the level of CD8⁺ cells of step aiv) to the level of CD4⁺ T cells of step aii); and

iv) the ratio of the level of CD14⁺CD16 monocytes of step av) to the level of CD14⁺CD16⁺ monocytes of step avi);

in said reference group of cerebrospinal fluid (CSF) samples;

c) calculating a group average for any one or all of the ratios of step b i) - a iv) being determined in said reference group of CSF samples; e) calculating the reciprocal value of each group average value of step c), thereby obtaining the adjustment factor.

[00163] Again, in some embodiments, the step of obtaining said detected cell ratios as defined above may be an optional step. Everything being defined above for said data processing system (00152]) may apply mutatis mutandis to the corresponding computer program as defined above.

[00164] According to the present invention a computer program as mentioned above may refer to a program listing written in a programming language to implement an algorithm, and to binary code loaded in a computer-based apparatus, encompassing the accompanying documentation. A computer program as defined above may include, but is not limited to any software or any downloadable internet link known to a person skilled in the art comprising instructions to cause the data processing system as defined elsewhere herein to execute the defined steps.

[00165] Also comprised is a computer-readable medium having stored thereon one of the computer programs as defined above (either as defined in [00158], [00160], or as defined in [00162]). A computer-readable medium having stored thereon all of the computer programs as defined above may also be comprised herein. As used herein, a computer-readable medium having stored thereon the computer program may include, but is not limited to, a USB stick, a disc, DVD, CD, CD-ROM.

Kit

[00166] Also comprised by the present invention is a kit comprising a fluorescently labeled binding partner for CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56 and CD138.

[00167] According to the present invention, the fluorescently labeled binding partner for CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56 and CD138 are preferably provided in one or more containers or vials in a kit (pharmaceutical pack), which may be associated with a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration or diagnostics.

[00168] Thus, the present invention may comprise a kit comprising one vial or container comprising all of the specific fluorescently labeled binding partners mentioned above. Put it differently, said kit may comprise a preparation comprising a mixture of all of the fluorescently labeled binding partners defined above.

[00169] Also comprised by the present invention, is a kit comprising one vial for each fluorescently labeled binding partner, thereby comprising nine vials: one vial comprising a binding partner for CD45, one vial comprising a binding partner for CD3, one vial comprising a binding partner for CD4, one vial comprising a binding partner for CD8, one vial comprising a binding partner for CD14, one vial comprising a binding partner for CD16, one vial comprising a binding partner for CD19, one vial comprising a binding partner for CD56, and one vial comprising a binding partner for CD138.

[00170] Further comprised by the present invention is a kit comprising at least one vial or container comprising a mixture of at least one fluorescently labeled binding partner for NK cells, at least one vial or container comprising a mixture of at least one fluorescently labeled binding partner for CD4+ T cells, at least one vial or container comprising a mixture of at least one fluorescently labeled binding partner for B cells, at least one vial or container comprising a mixture of at least one fluorescently labeled binding partner for CD8+ T cells, at least one vial or container comprising a mixture of at least one fluorescently labeled binding partner for classical monocytes and at least one vial or container comprising a mixture of at least one fluorescently labeled binding partner for non-classical monocytes.

[00171] In some embodiments, the kit of the present invention may comprise at least one vial or container comprising a mixture of a fluorescently labeled binding partner for CD45, CD16 and/or CD56. A kit comprising at least one vial or container comprising a mixture of a fluorescently labeled binding partner for CD45, CD3 and/or CD4 may also be envisaged. A kit comprising at least one vial or container comprising a mixture of a fluorescently labeled binding partner for CD45, CD3 and/or CD8 may also be envisaged. The present invention may also comprise a kit comprising at least one vial or container comprising a mixture of a fluorescently labeled binding partner for CD45, CD19 and/or CD138. A kit comprising at least one vial or container comprising a mixture of a fluorescently labeled binding partner for CD45, CD14 and/or CD16 may also be envisaged.

[00172] According to the present invention, the kit may further comprise a carrier. In some embodiments, said carrier may also be comprised in one or more containers or vials comprising a binding partner for CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and CD138 as defined above.

[00173] The term "carrier" refers to a diluent, adjuvant, or vehicle with which the binding partner is compounded / mixed in the kit. Such carriers can be sterile liquids, such as water, buffer including PBS and oils including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or PBS is a preferred carrier.

Agent

[00174] Additionally, the present invention comprises an agent for use in the treatment of MS in a subject of the present invention, wherein the subject has been diagnosed as having MS according to the method of the present invention, wherein the agent is selected from the group consisting of Interferon-beta 1 a, Interferon-beta 1 b, Glatiramer acetate, Teriflunomide, Fingolimod, Dimethyl fumarate, Alemtuzumab, Ocrelizumab, Natalizumab, Cladribine, Daclizumab, Siponimod, Ofatumumab, high dose biotin, Mitoxantrone, Cyclophosphamide, Azathioprine.

[00175] The group of interferon-beta may include, but are not limited to, Betaferon (Betaserone in the US), Rebif, Extavia, Avonex or Plegridy, whereas Betaferon, Extavia belong to the group of interferon-beta 1 b and whereas Rebif, Avonex and Plegridy belong to the group of interferon- beta 1 a.

[00176] According to Wiendl 2017, Nature Reviews, vol. 13, pp. 573-574 different MS measures are of importance for the right treatment concepts using the correct agent for the treatment of MS.

[00177] A dose of a therapeutically effective amount of the agent of the present invention may be administered to a subject in need thereof. How often a dose of a therapeutically effective amount of said agent may be administered to a subject may depend on the agent being used. MS treatment is often a long-term treatment, such as for 10-15 years, in some cases even for 25 years. For such long time periods, for example Interferon-betas, Glatiramer acetate, Natalizumab or Fingolimod may be given. For other agents being defined above, maximum treatment durations may be in the range of years, not decades (e.g., for Ocrelizumab once every six months, for Mitoxantrone once every three months).

[00178] As used herein, a "therapeutically effective amount" refers to an amount of the therapeutic active component or agent which is sufficient to treat or ameliorate a disease or disorder, to delay the onset of a disease or provides any therapeutical benefit in the treatment or management of a disease. A certain dose of a therapeutically effective amount of said agent may be known to a person skilled in the art and may vary depending on the specific agent being used. In general, a dose of a therapeutically effective amount of said agent may be in the range between about 20 pg to about 1000mg.

[00179] Further, said administration of (a dose of a therapeutically effective amount of) the agent of the present invention may be performed by injection or by infusion. The agent may be injected. This injection may be performed intraperitoneally, intravenously, subcutaneously, intrathecally (i.e. into the cerebrospinal fluid) or intramuscularly. In this context, the term “injection” refers to the administration of a liquid comprising for example said agent by applying a syringe and a hollow needle, which is pierced though the skin to be administered into the body.

[00180] A dose of a therapeutically effective amount of the agent may also be infused. In this context, the term“infusion” refers to a continuous, most commonly parenteral administration of liquid comprising for example the agent intravenously.

[00181] A dose of a therapeutically effective amount of the agent of the present invention may also be taken orally. The oral administration refers to swallowing said agent of the present invention with water or any other liquid used as a pharmaceutically acceptable carrier.

[00182] Thus, the present invention may comprise that (a dose of a therapeutically effective amount of) the agent of the present invention may be administered intraperitoneally, intravenously, subcutaneously, intramuscularly or orally. Preferably, said agent of the present invention may be administered orally.

EXAMPLES OF THE INVENTION

The following Examples illustrate the invention, but are not to be construed as limiting the scope of the invention.

Material and Methods

[00183] Patient recruiting and inclusion.

[00184] A total of 26 treatment-naive patients with MS, also including clinically isolated syndrome (CIS) diagnosed based on established criteria receiving an LP for diagnostic purposes, were prospectively recruited for this study (Tab. 2).

[00185] The control group consisted of 22 patients diagnosed with idiopathic intracranial hypertension (IIH) (Tab. 2). Patients were recruited and processed in three consecutive cohorts. CSF cells from cohort 1 were used for unsorted single cell RNA-seq (6 IIH vs. 6 MS patients). CSF cells from cohort 2 were analysed by flow cytometry only (7 IIH vs. 11 MS patients), and from cohort 3 were flow sorted for RNA-seq of CD3⁺CD4⁺CXCR5⁺ TFH cells (9 IIH vs. 9 MS patients). Patient details are provided in Table 2 and Figure 6. All patients gave written informed consent. The study was performed in accordance with the declaration of Helsinki and approved by the local ethics committees.

[00186] For MS patients, formal inclusion criteria were defined as: 1 ) treatment naive patients with a first episode suggestive of MS (i.e. clinically isolated syndrome (CIS)) or with relapsing- remitting (RR)MS diagnosed based on MAGNIMS criteria (Filippi et al. 2016, Lancet Neurol. 15, 292-303; Montalban et al. 2010, Neurology 74, 427-34) 2) patients receiving lumbar puncture for diagnostic purposes and consenting into participating in the study. Exclusion criteria for MS patients were defined as: 1 ) questionable diagnosis of MS by clinical signs or magnetic resonance imaging (MRI) findings, 2) secondary chronic progressive MS or primary progressive MS. IIH patients were included, if they gave informed consent. Exclusion criteria for all patients were: 1 ) immunologically relevant co-morbidities (e.g. rheumatologic diseases), 2) severe concomitant infectious diseases (e.g. HIV, meningitis, encephalitis), 3) pregnancy or breastfeeding, 4) younger than 18 years, 5) mental illness impairing the ability to give informed consent, 6) artificial blood contamination during the lumbar puncture resulting in >200 red blood cells / mI in routine CSF analysis. MS patients whose diagnostic work-up revealed a diagnosis other than MS within four weeks of clinical follow-up were retrospectively excluded from the study. The recruitment algorithm is illustrated in Figure 6C.

[00187] Diagnostic tests were performed in all patients screened as MS patients to exclude differential diagnoses. Specifically, the following tests were performed: PCR specific for CMV, EBV, HHV-6, HSV-1 , HSV-2 and VZV in CSF. Blood tests for anti-HAV IgM, HBsAg, anti-HBc, anti-HCV, rheuma factor, Waaler-Rose Test, anti cyclic citrullinated peptide (CCP), antinuclear antibody (ANA), anti-dsDNA, antineutrophil cytoplasmic antibodies (ANCA). CSF and serum were tested by the Treponema pallidum hemagglutination assay (TPHA) and a chemiluminescence assay (CLIA). For Borrelia burgdorferi detection CSF and blood were tested with an ELISA.

Table 2: Summarized information about patients in the present study.

Clinical characteristics (age, sex) of all control (IIH, n=22) and multiple sclerosis (MS, n=26) patients included in the study after screening are depicted. Numbers of excluded patients for each group were also shown. All included patients were divided over three cohorts, cohort 1: CSF samples used for single cell RNA-seq. (6 control vs. 6 MS), cohort 2: CSF samples analysed by flow cytometry only (7 control vs. 11 MS) and cohort 3: CSF samples flow sorted for RNA-seq of CD3⁺CD4⁺CXCR5⁺ TFH cells (9 control vs. 9 MS). Average ±SD

[00188] Sampling and flow cytometry analysis of cerebrospinal fluid cells.

[00189] Lumbar punctures were performed under sterile conditions using 20G Sprotte Canulae (Pajunk Medical). Up to 5 ml of CSF and 3 ml of blood were collected for scientific purposes in this study, in addition to diagnostic material. All samples were pseudonymised at collection. CSF was transported to further processing as quickly as possible. CSF samples were then centrifuged at 300g for 10 min. The supernatant was removed and CSF cells were resuspended in 5 ml of X-Vivo 15 media (Lonza) and subsequently stored at 4°C until further processing, to limit cell death and transcriptional changes. For CSF flow cytometry, cells were incubated in Versa Lyse buffer (Beckman Coulter). Afterwards, Cells were stained using following antibodies from Beckman Coulter: anti-human CD3 Pe-Cy5.5; anti-human CD4 APC; anti-human CD8 PB; anti-human CD14 FITC; anti-human CD16 AF750; anti-human CD19 AF700; anti-human CD45 KrO; anti-human CD56 Pe-Cy7; anti-human CD138 Pe; anti-human H LA-DR ECD and analyzed using a flow cytometer (Navios, Beckman Coulter). For scRNA-seq, CSF cells in media were centrifuged at 400 g for 5 min and resuspended in 40 mI of X-Vivo medium. 5 mI of the single cell suspension was stained with Trypan blue at a 1 :1 ratio and live cells were manually counted in a Fuchs-Rosenthal chamber. The maximum of CSF cells used for input was 10,000 cells. If total available CSF cell numbers were lower than 10,000 cells, all available cells were processed. On average 5,917 cells ± 1 ,505 SD (control 6,167 cells ± 2,614 SD vs. MS 5,667 cells ± 1 ,506 SD) CSF cells were used per donor.

[00190] Calculation of composite score.

[00191] A summed composite score differentiating flow cytometry results of MS from control patients was calculated. First, for each sample analysed by flow cytometry four ratios were calculated: 1 ) the ratio of the proportion of NK to CD4⁺ T cells, 2) the ratio of B to CD4⁺ T cells, 3) the ratio of CD8⁺ to CD4⁺ T cells, and 4) the ratio of CD14⁺CD16 to CD14⁺CD16⁺ monocytes. Each resulting group average was normalized to a value of 1 by dividing individual values by the group average. These four normalized ratios were added to obtain a basic composite score. A value of 1 was added to the basic composite in each case that an elevated immunoglobulin index or oligoclonal bands were detected in the samples; i.e. a maximum of 2 was added to score. This extended composite score was named 'Munster MS composite’ score. Receiver operator curve (ROC) analysis of the composites was performed and the area under the curve (AUC) was calculated using the Glm and rocplot functions of the Deducer package vO.7-9 in R.

[00192] Generation of single cell libraries and sequencing.

[00193] Single cell suspensions were loaded onto the Chromium Single Cell Controller using the Chromium Single Cell 3' Library & Gel Bead Kit v2 (both from 10X Genomics) chemistry following the manufacturer’s instructions. Sample processing and library preparation was performed according to manufacturer instructions using AMPure beads (Beckman Coulter). Sequencing was carried out on a local lllumina Nextseq 500 using the High-Out 75 cycle kit with a 26-8-0-57 read setup. Average sequencing depth was 51 ,064 ± 13,041 SEM (56,693 cells ± 46,649 cells SD) reads/cells (Table 3).

Table 3: Flow sorting related information.

[00194] Preprocessing of sequencing data. [00195] Processing of sequencing data was performed with the cellranger pipeline v2.0.2 (10X Genomics) and according to the manufacturer’s instructions. Raw bcl files were de-multiplexed using the cellranger mkfastq pipeline. Subsequent read alignments and transcript counting was done individually for each sample using the cellranger count pipeline with standard parameters. The cellranger aggr pipeline was employed, to ensure that all samples have the same number of confidently mapped reads per cell. The cellranger computations were carried at the High Performance Computing Facility of the Westfalische Wilhems-University (WWU) Munster. The pre-quality control (QC) total cell number was 26,819 with an average of 2,992.6 ± 1 ,343.87 SEM control-derived and 2,371.2 ± 565.5 SEM MS-derived cells used for analysis. This resulted in an average cell recovery rate of 47% ± 9% SEM compared to input (control 45% ± 15% vs MS 49% ± 13%)

[00196] Clustering and differential expression analysis.

[00197] Subsequent analysis steps were carried out with the R-package Seurat v2.1 (Satija et al. 2015, Nat. Biotechnol. 33, 495-502) using R v3.4.2 and RStudio v1.0.136. In order to account for differences in the total number of molecules per cell, the UMI data was normalized to a total of 10⁴ molecules and then transformed into log-space. After loading the dataset, doublets and low quality cells were removed. Specifically, doublet removal was performed in a cell type specific manner. For this, cells were preliminary clustered and the resulting Seurat object was then subset based on cluster identities. All cells with a >2.5-fold difference in numbers of UMIs/genes detected compared to the average level within their respective cluster were removed from each cluster. The object was merged back together and all cells with more than 5% mitochondrial genes were removed, as these cells most likely represent low quality cells. After QC the total remaining cell number was 25,835 with an average of 2,915.6 ± 1 ,326.5 SEM control and 2,251.4 ± 550.4 SEM MS cells used for further analysis.

[00198] The MeanVarPlot function from Seurat was then used to identify the top -2,000 genes displaying the highest variance within the dataset. Dimensionality reduction was done by Principal Component analysis (PCA), using the identified high variance genes as input. Statistically significant Principal Components (PCs) were identified by a combination of a JackStraw significance test and plotting Standard Deviations of the PCs. Cell Clustering was based on a graph-based clustering approach, employing K-nearest neighbour, and then visualized by t-distributed stochastic neighbour embedding (t-SNE). Differentially expressed genes were identified by a tobit-censoring model for zero inflated data (Satija et al. 2015, Nat. Biotechnol. 33, 495-502).

[00199] Bulk RNA-Seq of sorted TFH cells. CSF TFH cells were sorted on a BD FACS Aria™ III cell sorter using FACS Diva™ software following manufacturer’s instructions using an 85 miti nozzle and the drop delay was determined using BD Accudrop™ beads. Sorting was performed using sort precision mode“purity” for live CD3⁺CD4⁺CXCR5⁺ cells. Antibodies against PD-1 (EH12.2H7) and ICOS (C398.4A) were from Biolegend. Cells were sorted directly into 1 ,5 ml reaction tubes containing 100 pi RNA Lysis Buffer (Zymo Research). After sorting, tubes were vortexed, briefly centrifuged and frozen at -80 °C until RNA isolation. Data were analyzed using FlowJo software v10.4.1 (Tree Star, Inc.). Samples for bulk RNA-sequencing were prepared using a modified version of the SmartSeq2 protocol (Picelli et al. 2014, Nat. Protoc. 9, 171 -181 ). Unquantified purified RNA was used as input. Reaction volumes were scaled up and the number of PCR cycles during cDNA amplification adjusted accounting for the higher number of input cells compared to the original protocol (Picelli et al. 2014, Nat. Protoc. 9, 171 -181 ). Library Preparation was done by the Next Ultrall FS DNA Library Prep Kit (New England Biolabs) using 1 -3 ng of cDNA as input. Sequencing for 9 MS samples and 9 IIH samples was carried out on a NextSeq500 using the High-Out 75 cycle kit (lllumina).

[00200] Bulk expression quantification.

[00201] RNA-seq reads were aligned to the RefSeq hg38 transcriptome (GRCh38.2) using Bowtie2 (Langmead et al., 2009). The resulting transcriptome alignments were processed using the RNA-Seq by Expectation Maximization (RSEM) toolkit to estimate expected counts over RefSeq transcripts (Li and Dewey, 201 1 ). Several genes were quantified multiple times due to alternative isoforms unrelated by RefSeq annotation. Before expression data normalization, the gene entry with maximum counts was selected to represent the gene in further analysis.

[00202] Bulk data filtering.

[00203] Sample and gene filtering were similar to the scRNA-seq filtering method above, enforcing (> 107k reads, > 10% read alignment (forced), > 93.3% common genes detected; corresponding to z_cut = 20). A total of 5 samples were removed, leaving 13 samples. Setting n_s = 1 , 1 1 ,383 genes below were analysed.

[00204] For each sample, transcriptome alignment and quality metrics using FastQC (Babraham Bioinformatics), Picard tools (Broad Institute), and custom scripts were computed. Computed metrics included: (1 ) number of reads; (2) number of aligned reads; (3) percentage of aligned reads; (4) number of duplicate reads; (5) primer sequence contamination; (6) average insert size; (7) variance of insert size; (8) sequence complexity; (9) percentage of unique reads; (10) ribosomal read fraction; (1 1 ) coding read fraction; (12) UTR read fraction; (13) intronic read fraction; (14) intergenic read fraction; (15) mRNA read fraction; (16) median coefficient of variation of coverage; (17) mean 5’ coverage bias; (18) mean 3’ coverage bias; and (19) mean 5’ to 3’ coverage bias.

[00205] Bulk data normalization, unsupervised, and supervised analysis.

[00206] Data were normalized using SCONE. 569 positive controls were derived from MSigDB C7 entries annotated to include TFH cell types, including the most frequently included gene symbols in those entries. Negative controls for RUVg and evaluation were derived from the housekeeping gene list. Control lists were sampled down to 186 genes per list so as to match mean expression of genes in each list. The study group included two batches with 4/3 and 3/3 MS/IIH samples respectively. Biological condition was used only for evaluation. SCONE recommended TMM scaling and adjustment for 2 factors of RUVg and batch condition.

[00207] PCA on the scaled log-transformed normalized data for visualization was performed. DE between MMS and IIH donors was performed with limma-voom, using RUVg factors and batch in the model to adjust for unwanted variation. Per-gene DE significance scores were computed from log-transformed P-values and used for GSEA enrichment testing. Sets considered for testing included numbers 3,5, and 6 described in the VISION section. The 42 most frequent core members of the significant enrichments (Bonferroni adjusted P-value less than 0.01 ) - genes driving 7 or more of these enrichments - were selected and their normalized log values were correlated against each-other and represented in a sorted heatmap using pheatmap defaults.

[00208] Expression deconvolution using scRNA-seq data.

[00209] Cibersort was used for RNA expression deconvolution (Newman et al. 2015, Nat. Methods 12, 453-7) on the E-MTAB69 dataset described previously (Brynedal et al. 2010, Neurobiol. Dis. 37, 613-621 ). A customized RNA signature was extracted based on the scRNA seq data (no quantile normalization, permutations 100, q-value 0.1 ). UMI were transformed for correlation with microarray expression (x=log2(y+2)^*1.5). Only correlations with p < 0.05 were used. The resulting signature contained 91 genes. A deconvolution of the original scRNAseq data served as control, and showed a specific detection of all cell types (> 0.90 pearson correlation).

[00210] Statistical analyses.

[00211] GraphPad Prism 5 was used for statistical analysis of all mouse-related data. Unless mentioned otherwise in the figure legend, Student’s t-test for unrelated samples was used to calculate significance and p-value<0.05 was considered significant. R version 3.4.4 and RStudio 1.1.447 were used for the analysis of clinical and human flow cytometry data. [00212] Example 1 : Single-cell transcriptomics identifies the composition of cerebrospinal fluid cells.

[00213] It has been speculated that CSF cells would reflect disease mechanisms in MS more adequately than commonly studied peripheral blood cells. Processing of primary human CSF cells (see Material and Methods) decaying rapidly in nutrient-poor CSF has been optimized and these cells have been analysed using 1 ) microfluidics-based single cell RNA-sequencing (scRNA-seq), 2) flow cytometry, and 3) bulk RNA-seq of sorted T follicular helper (TFFI) cells (Fig. 1A). First scRNA-seq of total unsorted CSF cells from treatment-naive patients (n = 6) with either a first episode indicative of MS (i.e. clinically isolated syndrome (CIS)) or a first diagnosis of relapsing-remitting MS has been performed. For simplicity, this cohort is referred to as MS (Methods). Patients with idiopathic intracranial hypertension (IIH) served as control (n = 6), because CSF itself is normal in IIH while the production and absorption of CSF are unbalanced. Both cohorts were well matched (Fig. 6A and Tab. 2). Standard CSF and disease parameters were either comparable between groups or exhibited known MS-associated changes (Fig. 6B and Tab. 4).

[00214] After quality control and removal of low quality samples (2 per group), scRNA-seq approach returned transcriptional information of 22,357 total CSF cells with an average of 833 ± 193 SD genes detected per cell from 4 control and 4 MS donors (Tab. 3). After normalization (see Material and Methods) unbiased cell type clustering identified a total of 10 CSF cell clusters (Fig. 1 B and C). CD4⁺ T cells did not reliably subset into known lineages and were therefore tentatively merged into one cluster. Cluster identities based on marker gene expression (Fig. 1 D and E) have been manually assigned. CSF cells featured a strong predominance of T cells (CD4⁺ > CD8⁺) over monocyte lineage cells, natural killer (NK) cells, dendritic cells (DC), and B lineage cells (Fig. 1 B and C). As expected, granulocytes, megakaryocytes, and non-hematopoietic cells (e.g. neurons, glia, ependymal cells) were not detected in the CSF (Fig. 1B). Simultaneous flow cytometry confirmed this unique composition of CSF leukocytes (Fig. 1 F and Fig. 8A). Thus, single cell transcriptomics reliably reconstructs the composition of primary human CSF cells.

Table 4: Standard CSF parameters and MS disease features of patients in the present study.

[00215] Example 2: Single-cell transcriptomics identifies an MS-specific composition of CSF leukocytes.

[00216] Next, the dataset for disease-specific differences in CSF cell composition has been analysed (Fig. 2A). In MS, non-classical monocytes were less abundant than classical monocytes (Fig. 2A-C) and an increased ratio of classical / non-classical monocytes was confirmed by flow cytometry (Fig. 8B). An expansion of NK cells, of naive B cells, and of class- switched late B lineage cells (named plasma cells) in MS has also been found (Fig. 2A-C) that was confirmed by flow cytometry (Fig. 8B-C). Of note, plasma cells were only detected in MS patients and were absent from control CSF (Fig. 2A). Overall, inter-donor variability was high (Fig. 8D) and while non-significant trends towards altered cell type abundance were observed (Fig. 8D and E). The proportion of CSF CD4⁺ T cells quantified by scRNA-seq and flow cytometry negatively correlated with myeloid lineage cells (Fig. 7) suggesting their reciprocal abundance.

[00217] Next, the goal was to better dissect the composition of CD4⁺ T cells in the scRNA-seq data. All cells assigned to the CD4⁺ cluster have been extracted, secondary normalization and clustering have been performed, and thereby eight sub-clusters have been identified (Fig. 2D and E). Two of these were ‘contaminating’ or remaining CD8⁺ T cells and monocytes not assigned correctly in the previous clustering. A transcriptionally very distinct (Fig. 2F) cluster of FOXP3 expressing (i.e. most likely regulatory (Treg)) T cells was more abundant in two of the MS donors (Fig. 7E) although differences did not reach group-wide significance (Fig. 2G). Clinical and MRI features were not different in these two patients supporting sub-clinical MS heterogeneity. Based on the expression of marker genes (Fig. 2F) and cluster specific gene set enrichment analysis (GSEA), two remaining clusters were transcriptionally best described as naive CD4 (SELL^hlCCR7^hlCD44^l0CD69^l0 and CD27^hl) and as early proliferating CD4 (SELL^hlCCR7^hlCD44^l0CD69^l0 and CD27'°). In accordance with an increased proliferative capacity, the proliferating CD4 cluster featured expression of ribosomal genes (e.g. RPS8, RPS6) and nucleus forming transcripts. Abundance of such proliferating T helper cells was significantly increased in MS-derived samples (Fig. 2G) potentially indicating local expansion of CD4 cells in the CSF in MS. Three clusters exhibited a memory-like phenotype and were transcriptionally best described as central memory CD4 (SELL^l0CCR7^l0CD69^hlCD44^hl and CD27^hi), as early effector memory CD4 (SELL^l0CCR7^l0CD69^hiCD44^hi and CD28^hi), and as late effector memory CD4 (SELL^l0CCR7^l0CD69^intCD44^hl and CD28'°) and showed no disease- specific changes in abundance. Flow cytometry detected no apparent differences in the proportion of total CD4⁺ vs. CD8⁺ T cells in MS (Fig. 2H) indicating that T cell changes are subtle in MS.

[00218] Next, the goal was to summarize MS-specific CSF cell changes and to test them for potential diagnostic potential. From the merged flow cytometry data (Fig. 8), proportion ratios of NK to CD4⁺ T cells, of B to CD4⁺ T cells, of CD8⁺ to CD4⁺ T cells, and of CD 14⁺CD 16 (classical) to CD14⁺CD16⁺ (non-classical) monocytes have been calculated and normalized the resulting group average. From the values a composite score that was higher in MS-derived than control CSF samples has been calculated (Fig. 9A) and in a receiver operator curve (ROC) analysis reached an area under the curve (AUC) of 0.8618 (Fig. 9B). When an elevated immunoglobulin index and oligoclonal bands has been merged into this score, its AUC increased to 0.9179 (Fig. 9C and D). This extended composite score (‘Munster MS composite’ score) thus discriminates MS with good sensitivity and specificity in this preliminary cohort and indicates that comprehensive characterization of CSF cells can propose novel diagnostic ideas.

[00219] Example 3: Identifying MS-specific transcriptional changes in CSF leukocytes.

[00220] Next, the data-set for MS-specific transcriptional changes has been queried in individual cell clusters. CD4⁺ T cell clusters exhibited an increased expression of MHC class II genes (e.g. HLA-DR) and of complement components (e.g. C1QB, C1QA) in MS patients indicating increased activation. In accordance, GSEA showed enrichment of metabolic pathways in MS. NK cells and CD8 cell clusters up-regulated genes associated with activation and cytotoxicity. CD8 cell clusters showed higher expression of GZMK, GZMA, CD74, and HLA genes. NK cells in MS-derived CSF showed higher expression of cytotoxic mediators ( PRF1 (encoding Perforin 1 ), GZMA, GZMB, GZMM, CTSW (encoding Cathepsin W)) and reduced expression of KLRB1 (encoding the inhibitory NK cell receptor CD161 ). GSEA in these cell types showed enrichment of translation pathways in MS consistent with activation. Overall, this suggests higher cytolytic capacity of cytotoxic CSF cells in MS. Monocyte clusters featured higher expression of genes associated with secretory activity (e.g. GRN encoding granulin) and were enriched for influenza infection-related gene-sets.

[00221] The mDC cluster showed an increased expression of MHC class II genes (e.g. HLA- DRA), of different Fc receptors ( FCER1G , FCGRT, FCER1A, FCGR2B) indicating activation and induction of CD1E, CD1C which may indicate increased lipid antigen presentation. Increased expression of CCL5 and CXCL16 may serve as chemoattractant for T cells and NKT cells in MS. The pDC cluster featured increased expression of IRF8, that controls several functional modules in differentiated pDCs and higher expression of MHC class I ( HLA-C ) and MHC class II genes ( HLA-DPA1 , HLA-DRB1) and of IL3RA (encoding CD123) indicating increased activation. pDCs also exhibited down-regulation of CXCR3 regulating migration. Further downregulated genes included CD4, ICAM3, CD48, LTB (lymphotoxin beta), TRBC1 (T cell receptor beta), IL2RG. Transcripts associated with APC function in pDC are thus downregulated in the CSF in MS. Plasma cells were not detected in control patients prohibiting calculation of differentially expressed genes. B cells upregulated markers of B cell differentiation (e.g. FCRLA, IRF8) in MS patients and showed increased expression of IGHM (encoding IgM heavy chain) indicative of an acute antibody response. In conclusion, different transcriptional changes reflect the ongoing immune cell activation in the CSF in MS. [00222] Example 4: Single cell transcriptomics can help interpreting MS genetics and available CSF cell profiles.

[00223] Next, the results have been compared systematically with available data-sets. The scRNA-seq data has been used to systematically infer the cellular composition of these unsorted CSF cells in MS relapse and remission patients (n = 26 per group) using a deconvolution algorithm (Newman et al. 2015, Nat. Methods 12, 453-7). An inferred increase of cells resembling plasma cells, CD8⁺ T cells, and proliferating CD4+ T cells in the CSF of MS patients has been found (Fig. 4A). First, this partly validates the findings in a larger and independent cohort of MS patients. Second, this demonstrates that tissue-specific scRNA-seq data can help understanding available patient-derived data in MS.

[00224] The immune cell type(s) causing or promoting MS remain subject of debate. Results from genome-wide association studies have been interpreted to reflect T cell-dependent mechanisms driving MS. -170 known genes associated with genetic MS risk loci have therefore been systematically evaluated against their respective mean expression levels in the CSF cell clusters being identified (Fig. 1). It has been found that a minority (17%) of MS risk genes were unspecifically expressed in multiple immune cell clusters in the data-set (e.g. CD58, CD28, TYK2) (Fig. 4B). Most MS risk genes were instead preferentially expressed in one or two CSF cell clusters. Such genes with a‘cluster-enriched’ pattern were mainly expressed in B cell and plasma cell clusters (19% of genes, e.g. CD40, CXCR5, BACH2), in NK cells (11 % of genes, e.g. MAPK1, TCF7, JAK1), in pDCs (9%, e.g. IKZF1, IRF8), and in monocyte and mDC cells (22%, e.g. CD86, IFNGR2). Notably, in CSF cells only 3% of MS risk genes showed highest expression in the CD4⁺ T cell cluster (e.g. FOXP1, SOCS1, IL7R) and 14% showed enrichment in CD8⁺ T cells (e.g. BATF, ETS1, IZKF3) (Fig. 4B). Although highest expression cannot be equated with highest functional relevance, the data suggest that multiple immune cell lineages in the CSF are affected by MS genetic risk. This argues for a multi-lineage immune etiology of MS.

[00225] Example 5: Class-switched B lineage cells accumulate in the CSF in MS

[00226] The expansion of B cells and presence of plasma cells in CSF was a uniquely MS- specific feature and such CSF B lineage cells have therefore been examined in greater detail. In the B cell cluster, IGHD (marker of naive B cells) was expressed in approximately 20% of B cells (Fig. 3A). The expression of heavy chains in the B cell cluster was IGHG1 > IGHG2 > IGHG3, while IGHG4 was absent and IGHM was diffusely expressed. Heavy chain expression in the plasma cell cluster was IGHG1 > IGHG3 > IGHG2 > IGHG4 and IGHA1 and IGHA2 (encoding IgA heavy chains) were expressed strongly in few plasma cells, respectively (Fig. 3B). This indicates that high affinity and high complement activating heavy chains were most abundant. IGKC (encoding the k-light chain) was expressed by the majority of plasma cells, while the l-light chains were far less abundant and were IGLC2 > IGLC3 > IGLC5 (Fig. 3B). IGLC1 , IGLC4 genes were not detected. This corresponds to a k-to-A ratio of 2.3 which is a normal range for blood B cells. When it has been analyzed which heavy and light chains were expressed highest on a per cell level, it has unexpectly been found an enrichment of IGHM in B cells and IGHG1 in plasma cells (Fig. 3D) while light chain usage was very closely correlated. This suggests that local B cell maturation and proliferation at least partially occur in the CSF compartment in MS.

[00227] Example 6: Expansion of B cell-helping TFH cells in the CSF in MS patients.

[00228] Based on these indicators of local B cell maturation, next it has been tested whether the T follicular helper (TFH) cell subset - a CD4⁺ T cell subset absolutely required for B cell- maturation - was present in the CSF. In fact, CD3⁺CD4⁺CXCR5⁺ TFH cells in the CSF have been identified (Fig. 5A). When their abundance in independent cohorts of patients has been quantified, a significant increase in the proportion of total TFH cells in MS has been found (Fig. 5B and Tab. 3). The proportion activated TFH cells expressing PD-1 and ICOS was also increased in MS (Fig. 5B) while the alternative CD4⁺CXCR5 PD-1 ⁺ subset was unchanged (data not shown). The abundance of activated TFH cells in the CSF positively correlated with the proportion of CSF plasma cells (Fig. 5C) suggesting that both subsets may be functionally related.

[00229] Next, CSF-resident TFH cells have been characterized in greater detail by performing bulk RNA-sequencing (see Material and Methods) of TFH cells sorted from the CSF of MS patients and controls. MS-specific transcriptional changes have been found in CSF-derived TFH cells to be comparably subtle (Fig. 5D). MS-derived TFH cells showed increased expression of CXCR4, that is associated with germinal center (GC) residency of TFH cells and migratory capacity to B cell areas. Expression of BATF - a key regulator of the GC reaction - was also increased in MS-derived TFH cells (Fig. 5D). In contrast, the negative TFH-regulator CTLA4 was also upregulated (Fig. 5D). These transcriptional changes may reflect an increased capacity of TFH cells in the CSF to migrate to B cell areas and induce B cell maturation. Expanded TFH cells in the CSF could thus contribute to local TFH/B cell interaction potentially driving CNS autoimmunity.

Claims

1. A method of diagnosing a subject with multiple sclerosis, comprising determining

i) the ratio of the level of NK cells to the level of CD4⁺ T cells;

ii) the ratio of the level of B cells to the level of CD4⁺ T cells;

iii) the ratio of the level of CD8⁺ cells to the level of CD4⁺ T cells, and

iv) the ratio of the level of CD14⁺CD16 monocytes to the level of CD14⁺CD16⁺ monocytes;

in a test cerebrospinal fluid (CSF) sample obtained from said subject, wherein the combination of said ratios i)-iv) is indicative for whether or not said subject suffers from multiple sclerosis.

2. The method of claim 1 , further comprising determining whether or not an elevated immunoglobulin index and/or oligoclonal bands can be detected in said test CSF sample.

3. The method of claim 1 or 2, wherein the level of NK cells, the level of CD4⁺ T cells, the level of B cells, the level of CD8⁺ cells, the level of CD14⁺CD16 monocytes and the level of CD14⁺CD16⁺ monocytes are detected using flow cytometry.

4. The method of any one of claims 1 -3, wherein detecting the level of NK cells comprises measuring CD45, CD16 and/or CD56.

5. The method of any one of claims 1 -4, wherein detecting the level of CD4⁺ T cells comprises measuring CD45, CD3 and/or CD4.

6. The method of any one of claims 1 -5, wherein detecting the level of B cells comprises measuring CD45, CD19 and/or CD138.

7. The method of any one of claims 1 -6, wherein detecting the level of CD8⁺ T cells comprises measuring CD45, CD3 and/or CD8.

8. The method of any one of claims 1 -7, wherein detecting the level of CD14⁺CD16 monocytes comprises measuring CD45, CD14 and/or CD16.

9. The method of any one of claims 1 -8, wherein detecting the level of CD14⁺CD16⁺ monocytes comprises measuring CD45, CD14 and/or CD16.

10. The method of any one of claims 1-9, wherein a binding partner for CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56 and/or CD138 is used.

11. The method of claim 10, wherein the binding partner is an immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like functions.

12. The method of any one of claims 1-1 1 , wherein said subject is a human.

13. The method of any one of claims 1 -12, wherein said subject is an adult.

14. The method of any one of claims 1 -13, wherein said subject is suspected to suffer from multiple sclerosis (MS).

15. The method of any one of the preceding claims, further comprising the steps of

a) multiplying

i) the ratio according to claim 1 i) with an adjustment factor of preferably about 18 to about 74,

ii) the ratio according to claim 1 ii) with an adjustment factor of preferably about 25 to about 101 ,

iii) the ratio according to claim 1 iii) with an adjustment factor of preferably about 2 to about 8,

iv) the ratio according to claim 1 iv) with an adjustment factor of preferably about 0.6 to about 2.3;

b) summing up each adjusted ratio of step a i) - a iv), thereby obtaining a basic composite score; optionally

c) adding a value of 1 to the basic composite score of step b) in each case if an elevated immunoglobulin index or oligoclonal bands has been detected in said test CSF sample, thereby obtaining an extended composite score.

16. The method of claim 15, further comprising comparing said basic composite score or said extended composite score determined in said test CSF sample with a reference value.

17. The method of claim 16, wherein if said basic composite score or said extended composite score is above the reference value, it is indicative for whether or not said subject suffers from multiple sclerosis.

18. The method of claim 16 or 17, wherein the reference value is at least about 2.5.

19. A method of determining an adjustment factor for the method of any one of claims 15-18, comprising the steps of

a) determining

i) the ratio of the level of NK cells to the level of CD4⁺ T cells,

ii) the ratio of the level of B cells to the level of CD4⁺ T cells,

iii) the ratio of the level of CD8⁺ cells to the level of CD4⁺ T cells, and/or iv) the ratio of the level of CD14⁺CD16 monocytes to the level of

CD14⁺CD16⁺ monocytes,

in a reference group of cerebrospinal fluid (CSF) samples comprising at least one sample obtained from a subject having multiple sclerosis and at least one sample obtained from a healthy subject;

b) calculating a group average for any one or all of the ratios of step a i) - a iv) being determined in said reference group of CSF samples;

c) calculating the reciprocal value of each group average value of step b), thereby obtaining the adjustment factor.

20. The method of claim 19, wherein the ratio of the number of samples obtained from a subject having multiple sclerosis to the number of samples obtained from a healthy subject comprised in the reference group is between about 30 % to about 70 % and about 70 % to about 30 %.

21. The method of claim 19 or 20, wherein the healthy subject has idiopathic intracranial hypertension (IIH).

22. A data processing system comprising a processor configured to perform a method comprising the steps of

a) obtaining

i) the detected level of NK cells,

ii) the detected level of CD4⁺ T cells,

iii) the detected level of B cells,

iv) the detected level of CD8⁺ cells,

v) the detected level of CD14⁺CD16 monocytes, and

vi) the detected level of CD14⁺CD16⁺ monocytes,

from a test CSF sample obtained from a subject;

b) determining

i) the ratio of the level of NK cells of step ai) to the level of CD4⁺ T cells of step aii),

ii) the ratio of the level of B cells of step aiii) to the level of CD4⁺ T cells of step aii),

iii) the ratio of the level of CD8⁺ cells of step aiv) to the level of CD4⁺ T cells of step aii), and

iv) the ratio of the level of CD14⁺CD16 monocytes of step av) to the level of CD14⁺CD16⁺ monocytes of step avi);

c) multiplying

i) the ratio of the level of NK cells to the level of CD4⁺ T cells with an adjustment factor of preferably about 18 to about 74,

ii) the ratio of the detected level of B cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 25 to about 101 , iii) the ratio of the detected level of CD8⁺ cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 2 to about 8, iv) the ratio of the detected level of CD14⁺CD16 monocytes to the detected level of CD14⁺CD16⁺ monocytes with an adjustment factor of preferably about 0.6 to about 2.3;

d) summing up each adjusted ratio of step c i) - c iv), thereby obtaining the basic composite score; optionally

e) adding a value of 1 to the basic composite score of step d) in each case if an elevated immunoglobulin index or oligoclonal bands has been detected in said test CSF sample, thereby obtaining an extended composite score; and f) comparing said basic composite score or said extended composite score determined in said test CSF sample with a reference value; and

g) indicating whether or not said subject suffers from multiple sclerosis.

23. A flow cytometry device capable of detecting

i) the level of NK cells;

ii) the level of CD4⁺ T cells;

iii) the level of B cells;

iv) the level of CD8⁺ cells;

v) the level of CD14⁺CD16 monocytes;

vi) the level of CD14⁺CD16⁺ monocytes;

in a test CSF sample obtained from a subject, comprising the data processing system of claim 22.

24. A computer program comprising instructions to cause the data processing system of claim 22 or the flow cytometry device of claim 23 to execute the steps of

a) obtaining

i) the detected level of NK cells, ii) the detected level of CD4⁺ T cells,

iii) the detected level of B cells,

iv) the detected level of CD8⁺ cells,

v) the detected level of CD14⁺CD16 monocytes, and

vi) the detected level of CD14⁺CD16⁺ monocytes,

from a test CSF sample obtained from a subject;

b) determining

i) the ratio of the level of NK cells of step ai) to the level of CD4⁺ T cells of step aii),

ii) the ratio of the level of B cells of step aiii) to the level of CD4⁺ T cells of step aii),

iii) the ratio of the level of CD8⁺ cells of step aiv) to the level of CD4⁺ T cells of step aii), and

iv) the ratio of the level of CD14⁺CD16 monocytes of step av) to the level of CD14⁺CD16⁺ monocytes of step avi);

c) multiplying

i) the ratio of the level of NK cells to the level of CD4⁺ T cells with an adjustment factor of preferably about 18 to about 74,

ii) the ratio of the detected level of B cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 25 to about 101 , iii) the ratio of the detected level of CD8⁺ cells to the detected level of CD4⁺ T cells with an adjustment factor of preferably about 2 to about 8, iv) the ratio of the detected level of CD14⁺CD16 monocytes to the detected level of CD14⁺CD16⁺ monocytes with an adjustment factor of preferably about 0.6 to about 2.3;

d) summing up each adjusted ratio of step c i) - c iv), thereby obtaining the basic composite score; optionally

e) adding a value of 1 to the basic composite score of step d) each case if an elevated immunoglobulin index or oligoclonal bands has been detected in said test CSF sample, thereby obtaining an extended composite score; and f) comparing said basic composite score or said extended composite score determined in said test CSF sample with a reference value; and

g) indicating whether or not said subject suffers from multiple sclerosis.

25. A computer-readable medium having stored thereon the computer program of claim 24.

26. A kit comprising a fluorescently labeled binding partner for CD45, CD3, CD4, CD8, CD14, CD16, CD19, CD56, and CD138.

27. The kit of claim 26, wherein the binding partner is an immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like functions.

28. An agent for use in the treatment of multiple sclerosis in a subject, wherein the subject has been diagnosed as having multiple sclerosis according to the method of any one of claims 1 -18, wherein the agent is selected from the group consisting of Interferon-beta 1 a, Interferon-beta 1 b, Glatiramer acetate, Teriflunomide, Fingolimod, Dimethyl fumarate, Alemtuzumab, Ocrelizumab, Natalizumab, Cladribine, Daclizumab, Siponimod, Ofatumumab, high dose biotin, Mitoxantrone, Cyclophosphamide, Azathioprine.