METHODS FOR DETECTING, ASSESSING SEVERITY AND TREATING MULTIPLE SCLEROSIS

Abstract

The present invention relates to methods for detecting, assessing severity and treating multiple sclerosis. The inventors showed an impairment of the function of CD8+ Treg cells in MS patients and they demonstrated here that several criteria correlated with the disease severity i.e. the percentage of CD8.sup.+CD45RC.sup.intCD161.sup.lowValpha7.sup. T cells in the blood, the secretion of IFNg and IL10 and the suppressive activity of the CD8.sup.+CD45RC.sup.intCD161.sup.lowValpha7.sup. T cells. In particular, the present invention relates to a method for determining whether a subject has or is at risk of having multiple sclerosis comprising i) determining the percentage of CD8.sup.+CD45RC.sup.intCD161.sup.lowValpha7.sup. T cells in a biological sample obtained from the subject, ii) comparing the percentage determined at step i) with a predetermined reference value and iii) detecting differential in the percentage determined at step i) with the predetermined reference value indicates that the subject has or is at risk of having multiple sclerosis.

Claims

1. A method for determining whether a subject has or is at risk of having multiple sclerosis and/or, if the subject has multiple sclerosis, assessing whether the subject has or may have a severe form of multiple sclerosis, comprising i) determining the percentage of CD8+CD45RC.sup.intCD161.sup.lowValpha7.sup. T cells in a biological sample obtained from the subject, ii) comparing the percentage determined at step i) with a predetermined reference value, wherein a differential between the percentage determined at step i) and the predetermined reference value indicates that the subject has or is at risk of having multiple sclerosis, or that the subject has a severe form of multiple sclerosis, and iii) administering a suitable treatment to a subject whose measurement is indicative of having or being at risk of having multiple sclerosis, or of having a severe form of multiple sclerosis.

2. (canceled)

3. A method for determining whether a subject has or is at risk of having multiple sclerosis and/or, if the subject has multiple sclerosis, assessing whether the subject has or may have a severe form of multiple sclerosis, comprising i) determining the production level of at least one cytokine by the population of CD8+CD45RC.sup.intCD161.sup.lowValpha7.sup. T cells in a biological sample obtained from the subject, ii) comparing the production level determined at step i) with a predetermined reference value, wherein a differential between the production level determined at step i) and the predetermined reference value indicates that the subject has or is at risk of having multiple sclerosis, or that the subject has a severe form of multiple sclerosis, and iii) administering a suitable treatment to a subject whose measurement is indicative of having or being at risk of having multiple sclerosis, or of having a severe form of multiple sclerosis.

4. (canceled)

5. The method of claim 3 wherein the cytokine is selected from the group consisting of IL-34, IFN, IL-2 and IL-10.

6. The method of claim 5 which comprises determining the production level of IFN.

7. The method of claim 5 which comprises determining the production level of IFN and IL-10.

8. A method for determining whether a subject has or is at risk of having multiple sclerosis and/or, if the subject has multiple sclerosis, assessing whether the subject has or may have a severe form of multiple sclerosis, comprising i) assessing the suppressive activity of the population of CD8+CD45RC.sup.intCD161.sup.lowValpha7.sup.T cells present in a biological sample obtained from the subject, ii) comparing the suppressive activity determined at step i) with a predetermined reference value, wherein a differential between the suppressive activity determined at step i) and the predetermined reference value indicates that the subject has or is at risk of having multiple sclerosis, or that the subject has a severe form of multiple sclerosis, and iii) administering a suitable treatment to a subject whose measurement is indicative of having or being at risk of having multiple sclerosis, or of having a severe form of multiple sclerosis.

9. (canceled)

10. The method of claim 1, wherein the biological sample is a blood sample, a cerebrospinal sample or a biopsy sample.

11. The method of claim 10 wherein the biological sample is a PBMC sample.

12. The method of claim 3, wherein the biological sample is a blood sample, a cerebrospinal sample or a biopsy sample.

13. The method of claim 12 wherein the biological sample is a PBMC sample.

14. The method of claim 1, wherein a percentage that is the same or higher than the corresponding predetermined reference value indicates that the disease is not severe; and the method includes a step of administering a suitable treatment to a subject whose measurement is indicative of having multiple sclerosis that is not severe.

15. The method of claim 3, wherein a production level that is the same or higher than the corresponding predetermined reference value indicates that the disease is not severe; and the method includes a step of administering a suitable treatment to a subject whose measurement is indicative of having multiple sclerosis that is not severe.

16. The method of claim 8, wherein the biological sample is a blood sample, a cerebrospinal sample or a biopsy sample.

17. The method of claim 16 wherein the biological sample is a PBMC sample.

18. The method of claim 8, wherein a suppressive activity that is the same or higher than the corresponding predetermined reference value indicates that the disease is not severe; and the method includes a step of administering a suitable treatment to a subject whose measurement is indicative of having multiple sclerosis that is not severe.

Description

FIGURES

[0040] FIG. 1: Percentage of peripheral human CD8.sup.+CD45RC.sup.int Tregs subsets in MS patients. CD8.sup.+CD45RC.sup.int T cells were analyzed in the blood of MS patients vs. healthy individuals for expression level of CD45RC.

[0041] FIG. 2: Percentage of peripheral blood and brain infiltrating human CD8.sup.+CD45RC.sup.int Tregs subsets in MS patients. CD8.sup.+CD45RC.sup.int T cells were analyzed in the blood and cerebrospinal fluids of MS patients for expression level of CD45RC.

[0042] FIG. 3: Percentage of human CD8.sup.+CD45RC.sup.low Tregs expressing IL-2, IFNg and/or IL10. CD8.sup.+CD45RC.sup.low T cells (discriminating CD45RC.sup.int and CD45RC.sup.neg) were analyzed in the blood of MS patients vs. healthy individuals for expression level of IL-2, IFNg and/or IL10.

[0043] FIG. 4: Percentage of CD8.sup.+CD45RC.sup.int Tregs expressing IFNg in MS patients with low or high MSSS. CD8.sup.+CD45RC.sup.int T cells were analyzed in the blood of MS patients for expression level of IFNg.

[0044] FIG. 5. Dose dependent inhibition of the proliferation of effector CD4.sup.+CD25.sup. T cells by CD8.sup.+CD45RC.sup.int. CFSE labelled recipient CD4.sup.+CD25.sup. effector T cells were mixed with allogeneic T-depleted PBMCs at a 1:1 ratio and CD3.sup.+CD161lowValpha7CD8.sup.+CD45RC.sup.int Tregs were added at different ratios. Proliferation of CD4.sup.+CD25.sup. T cells was analysed 5 days later by flow cytometry. upper panel represents suppressive capacity in MS patients with a high MSSS number (>3) and lower panel represents suppressive capacity of MS patients with a low MSSS number (<3) compared to healthy individuals. * p<0.05; ** p<0.01; Two way anova RM, Bonferroni post test.

[0045] FIG. 6. Dose dependent inhibition of the proliferation of effector CD4.sup.+CD25.sup. T cells by CD8.sup.+CD45RC.sup.int. CFSE labelled recipient CD4.sup.+CD25.sup. effector T cells were mixed with allogeneic T-depleted PBMCs at a 1:1 ratio and CD3+CD161lowValpha7-CD8.sup.+CD45RC.sup.low/neg/int Tregs were added at different ratios. Proliferation of CD4.sup.+CD25.sup. T cells was analysed 5 days later by flow cytometry. upper panel represent suppressive capacity in MS patients with a high MSSS number (>3) compared with MS patients with a low MSSS number (<3). lower panel represent suppressive capacity in function of MSSS number at effector: stimulator: suppressor ratio 4:4:1 (left) and 2:2:1 (right). Two Way RM ANOVA, Bonferroni post test, * p<0.05; ** p<0.01;*** p<0.001.

[0046] FIG. 7. Gating strategy to identify CD8.sup.+CD45RClowCD161-Va7.sup. Tregs.

EXAMPLE 1

[0047] Material & Methods

[0048] Healthy volunteers blood collection and PBMC separation: Blood was collected from healthy donors at the Etablissement Francais du Sang (Nantes, France). Approval for this study was obtained from the institutional review boards. Written informed consent was provided according to institutional guidelines. Blood was diluted 2-fold with PBS before PBMC were isolated by Ficoll-Paque density-gradient centrifugation (Eurobio, Courtaboeuf, 10 France) at 2000 rpm for 20 at room temperature without braking. Collected PBMC were washed in 50 mL PBS at 1800 rpm for 10 min and remaining red cells and platelets are eliminated after incubation 5 min in a hypotonic solution and centrifugation at 1000 rpm for 10 min. When indicated, PBMCs were frozen in DMSO:SVF 1:9 and washed twice in medium-10% SVF for thawing.

[0049] Cell Isolation:

[0050] T cells were obtained from PBMCs by negative selection by elutriation (DTC Plateforme, Nantes) and magnetic depletion (Dynabeads, Invitrogen) of B cells (CD19+ cells) and remaining monocytes (CD14+ and CD16+ cells) before sorting of CD3+CD4+CD25 cells as responder cells, CD3+CD161.sup.lowValpha7.sup.CD4.sup.CD45RC.sup.low (int or neg) as CD8+ Tregs. Tregs sorted from thawed PBMCs were stimulated 24 h with anti-CD3 and anti CD28 mAb (1 g/ml each) in presence of 250 U/ml IL-2 before plating. A FACS ARIA I (BD Biosciences, Mountain View, Calif.) was used to sort cells. APCs used as stimulator cells were obtained by magnetic depletion of CD3+ cells and 35Gy irradiation.

[0051] Monoclonal Antibodies and Flow Cytometry:

[0052] For interleukins and IFNg analysis, PBMCs were stimulated with PMA (50 ng/ml) and ionomycine (1 g/ml) for 7 h in presence of Brefeldine A (10 g/ml) for the last 4 h. Fluorescence was measured with a LSR II or a Canto II cytometer (BD Biosciences, Mountain View, Calif.) for phenotype and functional analysis respectively, and the FLOWJO software (Tree Star, Inc. USA) was used to analyze data. Cells were first gated by their morphology excluding dead cells by selecting viable cells.

[0053] Mixed Lymphocyte Reaction:

[0054] Tregs suppressive activity was assessed on syngeneic effector CD4+CD25T cells stimulated with allogeneic APCs. Experiments were realized with 1:1 APCs: responder ratio. Proliferation of CFSE-labeled responder cells was analyzed by flow cytometry after 4.5 days of coculture in 5% AB serum medium, by gating on CD3+CD4+ living cells (DAPI negative).

[0055] Results

[0056] Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the Central Nervous System (CNS), probably of autoimmune origin. Autoimmune diseases can develop following pathological activation of autoreactive effector cells and/or, alternatively, after weakening of self-protective regulatory mechanisms, for example regulatory T cells (Tregs). While most of the studies about the role of Tregs in autoimmunity have focused on CD4.sup.+ T cells, the role of CD8.sup.+ Tregs in MS remains largely unexplored. It has been shown that some subsets of CD8.sup.+ T cells, including CD8.sup.+ Qa-1.sup.+, CD8.sup.+CD28.sup. and CD8.sup.+CD122.sup.+ T cells, can have a regulatory role in Experimental Autoimmune Encephalomyelitis (EAE), the mouse model of MS (Hu D et al., 2004; Najafian N et al., 2003; Yu P et al., 2014). However, in human, CD8.sup.+ Tregs are less described.

[0057] Recently, we have described a new subset of regulatory CD8.sup.+CD45RC.sup.low Treg subpopulation, able to produce IFN, IL10 and IL34 and displaying a great efficiency in regulating Graft vs Host Disease (GVHD) and human skin transplantation rejection in humanized mice (Picarda et al, JCI, 2014 and Bezie et al, JCI, 2015, Bzie et al., in preparation). The aim of our study is to analyze the frequency and function of this particular subset of CD8.sup.+ T cells in the blood of MS patients compared to Healthy Volunteers (HV) and in cerebrospinal fluids of MS patients.

[0058] We have recruited age- and gender-matched 22 MS patients and 24 healthy volunteers. Inclusion criteria were as follows: age between 18 and 55, relapsing MS, EDSS between 0-5.5, immunomodulatory treatment stopped at least 6 months ago, immunosupressant treatment stopped at least 12 months ago, natalizumab stopped at least three months ago. Importantly, all our analysis excluded MAIT cells (CD8.sup.+CD161.sup.highValpha7.sup.+ cells) that may have a role in MS. In addition, we discriminated in all our analysis CD45RC.sup.high (data not shown), CD45RC.sup.int and CD45RC.sup.neg.

[0059] Analysis of the CD8.sup.+CD161.sup.lowValpha7-CD3.sup.+CD45RC subpopulations of cells in MS patients versus healthy volunteers showed a trend for a decreased in the percentage of CD8.sup.+CD45RC.sup.intCD161.sup.lowValpha7-CD3.sup.+ cells and no differences for the other subsets (FIG. 1A and data not shown). Interestingly, a more precise analysis showed a significant increase of the frequency of CD8.sup.+CD45RC.sup.intCD161.sup.lowValpha7.sup.CD3.sup.+ but not CD8.sup.+CD45RC.sup.negCD161.sup.lowValpha7.sup.CD3.sup.+ T or CD8.sup.+CD45RC.sup.highCD161.sup.lowValpha7.sup.CD3.sup.+ T cells in the blood of MS patients with a low MSSS compared to HV and MS patients with a high MSSS, demonstrating that an increased frequency of CD8.sup.+CD45RC.sup.intCD161.sup.lowValpha7.sup. CD3.sup.+ Tregs correlated with a lower disease (FIG. 1B and data not shown).

[0060] Analysis in MS patients in the blood versus the cerebrospinal fluid (CSF) demonstrated significantly more CD8.sup.+CD45RC.sup.negCD161.sup.lowValpha7.sup. CD3.sup.+ T cells but similar levels of CD8.sup.+CD45RC.sup.intCD161.sup.lowValpha7.sup.CD3.sup.+ T cells in the CSF of MS patients compared to the blood (FIG. 2).

[0061] Analysis of cytokine secretion capacity upon short-term stimulation demonstrated that a significant decrease in production of IFN, IL-2 and IL-10/IFN by CD8.sup.+CD45RC.sup.intCD161.sup.lowValpha7.sup.CD3.sup.+ T cells from MS patients compared to controls (FIG. 3). Further analysis revealed that the altered IFNg secretion profile in CD8.sup.+CD45RC.sup.intCD161.sup.lowValpha7.sup.CD3.sup.+ T cells in the blood of MS patients correlated with a low severity score versus healthy volunteers and MS patients with a high severity score (FIG. 4).

[0062] Next, CD8.sup.+CD45RC.sup.int, neg or high CD161.sup.lowValpha7.sup.CD3.sup.+T cells from MS or HV individuals were evaluated for their suppressive activity on proliferation of syngeneic effector CD4.sup.+CD25.sup.T cells stimulated with allogeneic T-depleted PBMCs, in an effector: suppressor dose dependent manner (FIG. 5). We observed that the suppressive capacity of CD8.sup.+CD45RC.sup.int CD161.sup.lowValpha7.sup.CD3.sup.+T cells from high MSSS patients was significantly altered while the one from low MSSS patients remained unchanged and similar to HV. In addition, we demonstrated that the suppressive activity of CD8.sup.+CD45RC.sup.intCD161.sup. T cells from MS patients correlated with the severity of the MS disease (FIG. 6).

[0063] Altogether, these results suggest an impairment of the function of CD8.sup.+ Treg cells in MS patients and we demonstrated here that several criteria correlated with the disease severity i.e. the percentage of CD8.sup.+CD45RC.sup.intCD161.sup. T cells in the blood, the secretion of IFNg and the suppressive activity of the CD8.sup.+CD45RC.sup.intCD161.sup. T cells.

[0064] This study suggests that CD8.sup.+CD45RC.sup.low and its subsets may be potential therapeutic and prognostic tools in MS patients, correlating with the progression of the disease.

EXAMPLE 2: MATERIAL AND METHODS OF RESULTS SHOWED IN FIG. 7

MLR:

[0065] PBMCs from MS patients and age and sexe-matched healthy volunteers were thawed and washed in medium, counted, and cell concentration was adjusted at 210.sup.8 PBMC/ml in PBS-FCS-EDTA. Cells were incubated with anti-CD3-Pe, anti CD4-PerCPCy5.5, anti-CD25-APC Cy7, anti CD161-APC, anti Valpha7.2 PeCy7 and anti-CD45RC FITC mAbs 30 4 C. Cells were washed with PBS-FCS-EDTA, filtered on 60 m tissue, labeled with Dapi and FACS Aria sorted on lymphocyte morphology, exclusion of doublet cells, and DAPI-CD3.sup.+CD4.sup. CD45RC.sup.low/int/neg CD161.sup.lowValpha7.2.sup. expression as Tregs and DAPI.sup.CD3.sup.+CD4.sup.+CD25.sup. expression as Teff responder cells. After sorting, Tregs were washed in medium and Teff were labeled with CFSE and washed in medium. APCs were obtained by CD3.sup.+ cells depletion and 35Gy irradiation from thawed PBMCs pooled from 3 allogeneic healthy volunteers. Cells were plated at 1:1 Teff:APCs ratio and a range of Teff:Tregs ratio, in RPMI 1640 medium supplemented with 5% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), and non-essential amino acids (1). After 5 days culture, Teff proliferation was analyzed by CFSE analysis in DAPI.sup.CD4.sup.+CD3.sup.+ T cells.

FACS Pheno:

[0066] PBMCs from MS patients and age and sexe-matched healthy volunteers were thawed and washed in medium. Cells were stimulated 7 h with PMA-ionomycin in presence of Brefeldine A for the last 4 h, in RPMI 1640 medium supplemented with 5% AB serum, Penicillin (100 U/ml), Streptomycin (0.1 mg/ml), Sodium pyruvate (1 mM), Glutamine (2 mM), Hepes Buffer (1 mM), and non-essential amino acids (1X). Cells were washed in PBS, dead cells were labeled with fixable viability dye yellow 30 4 C. Cells were washed in PBS-FCS-EDTA, and stained with anti-CD161, anti-Valpha7.2, anti-CD8 and anti-CD45RC mAbs for 30 4 C. Cells were washed twice in PBS-FCS-EDTA, and permeabilized with Fixation-permeabilization kit (Ebiosciences) 30 4 C. Cells were washed in permeabilization buffer (Ebiosciences) and stained with anti-IFNg, anti-IL-34, and anti-IL-10 mAbs for 30 RT. Cells were washed twice in permeabilization buffer, once in PBS-FCS-EDTA and fixed in PBS-PFA2% 20 4 C. LSR II and Flowjo were used to analyze cytokines expression in Yellow CD8.sup.+CD45RC.sup.lowCD161.sup.lowValpha7.2.sup. cells.

REFERENCES

[0067] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.