WO2018029336A1 - Methods for determining whether a subject was administered with an activator of the ppar beta/delta pathway. - Google Patents

Abstract

The inventors have investigated the effect of overexpressing PPARβ on T cell biology in vivo by using mice that overexpress PPARβ in a T cell specific manner. Using this transgenic mouse model, and also by systemic treatment of wild-type mice with a PPARβ agonist, they have demonstrated that activation/overexpression of PPARβ increases the fatty acid oxidation capacity of developing T cells, thereby inhibiting the proliferative burst at the DN4 stage. Accordingly, he present invention relates to a method for identifying whether a subject was administered with an activator of the PPARβ/δ pathway comprising: i) quantifying the expression level of Acaa2, Acadvl and Cpt1a in a blood sample obtained from said subject; ii) comparing the expression determined at step i) with a predetermined reference value, and iii) concluding that the subject was administered with an activator of the PPARβ/δ pathway when the expression level of Acaa2, Acadvl and Cpt1a determined at step i) is higher than the predetermined reference value.

Description

METHODS FOR DETERMINING WHETHER A SUBJECT WAS ADMINISTERED WITH AN ACTIVATOR OF THE PPAR BETA /DELTA PATHWAY

FIELD OF THE INVENTION:

The invention is in the field of immunology, particularly, the invention relates to methods for determining whether a subject was administered with an activator of the PPAR β/δ pathway.

BACKGROUND OF THE INVENTION:

Recent studies have demonstrated the importance of metabolism in T cell biology and how metabolic changes drive T cell differentiation and fate (for recent reviews see (1-3)). More specifically, na^'ive T cells have a metabolically quiescent phenotype and use glucose, fatty acids, and amino acids to fuel oxidative phosphorylation to generate energy. Upon activation, quiescent na^'ive T cells undergo a rapid proliferation phase which is associated with dramatically increased bioenergetic and biosynthetic demands. To comply with these demands, activated T cells use aerobic glycolysis. At the conclusion of an immune response, decreased glycolysis and increased lipid oxidation can favor the enrichment of long-lived CD8+ memory cells. Furthermore, different T cell subsets have different metabolic signatures. Indeed, whereas effector T cells are highly glycolytic, regulatory T cells have high lipid oxidation rates. It was demonstrated that by directly manipulating T-cell metabolism one can regulate T cell fate, suggesting that it may be possible to target metabolic pathways and mediators to control the formation of T-cell lineages or to suppress T-cell responses by blocking specific metabolic pathways essential for T-cell growth and proliferation (4, 5). Normally, committed lymphoid progenitors arise in the bone marrow and migrate to the thymus (for review on T cell development see (6)). Thymocytes that express TCRs that bind self-peptide-MHC-class-I complexes become CD8+ single positive (SP) T cells, whereas those that express TCRs that bind self-peptide- MHC-class-II ligands become CD4+ SP T cells (γδ T cells are not MHC restricted); these cells are then ready for export from the medulla to peripheral lymphoid sites. In mice, DN4 thymocytes that have undergone a productive TCRP rearrangement show a proliferative burst (7). It is also during this stage that expression of the glucose transporter Glut-1 is highest, suggesting a high rate of glycolysis during this highly proliferative stage of T cell development (8). Inhibiting glycolysis by knocking out the glucose transporter Glut-1 during DN3/DN4 stages of T cell development leads to a disruption in T cell development at the DN4 stage (8). Peroxisome proliferator-activated receptor β (PPARP) is a ligand-activated transcription factor that belongs to the nuclear hormone receptor superfamily and plays an important role in the regulation of different physiological functions such as development, energy metabolism, cellular differentiation/proliferation, and inflammation (for a recent extensive review see (9)). We have previously shown that PPARP controls in myotubes, the expression of genes implicated in fatty acid (FA) uptake, handling and catabolism (Fatty Acid Translocase, FAT/CD36; Pyruvate dehydrogenase kinase 4, PDK4; and carnitine palmitoyltransferase 1A, CPT1A) and that in skeletal muscle, PPARP is upregulated in physiological situations characterized by increased lipido -oxidative metabolism, such as fasting or aerobic exercise training (10-12). These observations suggest that PPARP plays a central role in the transition of skeletal muscle fuel preference towards lipids during metabolic challenges where glucose oxidation needs to be limited. PPARp is present at the mRNA level in human CD4 and CD8+ T-cells and CD19+ B cells (13), and in peripheral blood T-cells (14). However there are no studies or proof in the art showing a role of PPAR family of nuclear receptors in regulating fuel preference in developing T cells. Thus, there is a need to understand the role of PPAR family on the regulation of the metabolism of T cells and their development.

SUMMARY OF THE INVENTION:

The present invention relates to a method for identifying whether a subject was administered with an activator of the PPARp/δ pathway comprising: i) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject; ii) comparing the expression determined at step i) with a predetermined reference value, and iii) concluding that the subject was administered with an activator of the PPARp/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step i) is higher than the predetermined reference value or concluding that the subject was not administered with an activator of the PPARp/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step i) is lower than the predetermined reference value. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors have investigated the effect of overexpressing PPARP on T cell biology in vivo by using mice that overexpress PPARP in a T cell specific manner. Using this transgenic mouse model, and also by systemic treatment of wild-type mice with a PPARP agonist, they have demonstrated that activation/overexpression of PPARP increases the fatty acid oxidation capacity of developing T cells, thereby inhibiting the proliferative burst at the DN4 stage. This leads to disruption of T cell development in the thymus with subsequent consequences for T cell populations in peripheral lymphoid organs. This study also suggests that stimulating lipid oxidation early on in T cell precursors will hamper their development into mature T cells. Furthermore, this only seems to affect αβ- but not γδ T cell development, suggesting that development of αβ-Τ cells is more easily affected by manipulating their metabolism. This is a first study demonstrating a direct role for a member of the PPAR family in T cell development.

Method for identifying whether a subject was administered with an activator of the ΡΡΑ β/δ pathway by quantifying the expression level of Acaa2, Acadvl and Cptl

Accordingly, in a first aspect, the invention relates to a method for identifying whether a subject was administered with an activator of the ΡΡΑΡνβ/δ pathway comprising: i) quantifying the expression level of Acaa2, Acadvl and Cptl a in a blood sample obtained from said subject; ii) comparing the expression determined at step i) with a predetermined reference value, and iii) concluding that the subject was administered with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptl a determined at step i) is higher than the predetermined reference value or concluding that the subject was not administered with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptl a determined at step i) is lower than the predetermined reference value. As used herein, the term "ΡΡΑΡνβ/δ" refers to Peroxisome proliferator-activated receptors (PPARs) which are a family of ligand-activated nuclear receptors that play key roles in the regulation of cellular differentiation, development, metabolism and inflammation. The three different PPAR isoforms (α, β or δ, and γ) exhibit tissue-selective expression and are activated by different physiological and synthetic activators. The major physiological functions of PPARs result from their activity as transcription factors, modulating the expression of specific target genes. PPARβ/δ is the predominant form in rodent and human skeletal muscle (SKM). Like other isoforms, PPARβ/δ controls transcription of several genes involved in metabolism, differentiation, inflammation, and proliferation. The term "PPARβ/δ pathway" refers to the signaling of molecules involved in the activation or inhibition of PPARβ/δ.

As used herein, the term "activator" refers to any compound, natural or not, that is able to increase the expression level of PPARβ/δ or activate the PPARβ/δ pathway. The activator can also act as an agonist. As used herein, the term "agonist" refers to any compound natural or not that is able to bind to PPARβ/δ and promotes PPARβ/δ biological activity. The terms "agonist" or "modulator" could be used interchangeably. According to the invention, the activators include but are not limited to peptides, polypeptides, protein, nucleic acids such as aptamers, small organic molecules (natural or not). Particularly, the agonist of ΡΡΑΡνβ/δ is a small molecule. The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. In a particular embodiment, the activator is a natural molecule such as unsaturated fatty acid; carbaprostacyclin or components of very low-density lipoprotein. In a particular embodiment, the activator is a synthetic molecule. In a particular embodiment, the activators of PPAR β/δ are described in WO 2009078981. In a particular embodiment, the activator is GW501516, also known as Endurobol, which is a selective activator of ΡΡΑΡνβ/δ receptor (Sznaidman et al., 2003). In a particular embodiment, the activator is MBX-8025. In some embodiments, the activator is KD0310. In some embodiments, the activator is GW0742 (also known as GW610742). In some embodiments, the activator is L- 165041 (Berger et al., 1999). In some embodiments, the activator of ΡΡΑΡνβ/δ is a-lipoic acid (a-LA). In some embodiments, the activator of ΡΡΑΡνβ/δ pathway is Enalaprilat, also known as Enalapril. In a particular embodiment, the activator of ΡΡΑΡνβ/δ pathway corresponds to a natural or synthetic inhibitor of the JNK pathway which increases the expression level of ΡΡΑΡνβ/δ. Accordingly, the activator of the ΡΡΑΡνβ/δ pathway is one of the compound as described in WO2010069833, WO2011151357, WO2011151358, WO2010097335, WO2010069833, WO2011151357, WO2008028860, WO2010046273, WO201213668, WO2013007676, WO2011071491, WO2006076595, WO2012145569, WO2004078756, WO2008095944, WO2010015803, WO2013074986, WO2013169793, WO2007125405, WO2012083092, WO2010108155, WO2001027268, WO2007031280, WO2011160653, WO2012048721, WO2013079213, WO2013091670, WO2010091310 or WO2014170706.

As used herein, the term "Acaa2" refers to acetyl-Coenzyme A acyltransferase 2, also known as 3-Ketoacyl-CoA thiolase is an enzyme that in humans is encoded by the ACAA2 gene and catalyzes the last step of the mitochondrial fatty acid beta oxidation spiral. The naturally occurring human Acaa2 gene has a nucleotide sequence as shown in Genbank Accession number NM 006111.2 and the naturally occurring human Acaa2 protein has an aminoacid sequence as shown in Genbank Accession number NP 006102.2. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NMJ77470.3 and NP_803421.1).

As used herein, the term "Acadvl" refers to very long-chain specific acyl-CoA dehydrogenase, is an enzyme that in humans encoded by the ACADVL gene and catalyzes most of fatty acid beta-oxidation by forming a C2-C3 trans-double bond in the fatty acid. The naturally occurring human Acadvl gene has a nucleotide sequence as shown in Genbank Accession number NM 000018.3 and the naturally occurring human Acadvl protein has an aminoacid sequence as shown in Genbank Accession number NP 000009.1. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM 017366.3 and NP_059062.1).

As used herein, the term "Cptla" refers to carnitine palmitoyltransferase 1A, is a mitochondrial enzyme responsible for the formation of acyl carnitines by catalyzing the transfer of the acyl group of a long-chain fatty acyl-CoA from coenzyme A to 1-carnitine. The naturally occurring human Cptla gene has a nucleotide sequence as shown in Genbank Accession number NM 001031847.2 and the naturally occurring human Cptl a protein has an aminoacid sequence as shown in Genbank Accession number NP 001027017.1. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM_013495.2 and NP_038523.2).

As used herein, the term "expression level" corresponds to the expression level of each of the 3 genes. Typically, the expression level of the 3 genes may be determined by any technology known by a person skilled in the art. In particular, each gene expression level may be measured at the genomic and/or nucleic and/or protein level. In a particular embodiment, the expression level of gene is determined by measuring the amount of nucleic acid transcripts of each gene. In another embodiment, the expression level is determined by measuring the amount of each gene corresponding protein. The amount of nucleic acid transcripts can be measured by any technology known by a man skilled in the art. In particular, the measure may be carried out directly on an extracted messenger RNA (mRNA) sample, or on retrotranscribed complementary DNA (cDNA) prepared from extracted mRNA by technologies well-known in the art. From the mRNA or cDNA sample, the amount of nucleic acid transcripts may be measured using any technology known by a man skilled in the art, including nucleic microarrays, quantitative PCR, microfluidic cards, and hybridization with a labelled probe. In a particular embodiment, the expression level is determined using quantitative PCR. Quantitative, or real-time, PCR is a well-known and easily available technology for those skilled in the art and does not need a precise description. Methods for determining the quantity of mR A are well known in the art. For example the nucleic acid contained in the biological sample is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR). Particularly, quantitative or semi-quantitative RT-PCR is performed. Real-time quantitative or semiquantitative RT-PCR is particularly advantageous.

Other methods of amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences. In a particular embodiment, the method of the invention comprises the steps of providing total R As extracted from a biological samples and subjecting the R As to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi-quantitative RT-PCR.

In another embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere- sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a biological sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200- 210).

As used herein, the term "subject" refers to any mammals, such as a rodent, a feline, a canine, an equidae and a primate. Particularly, in the present invention, the subject is a human. In a particular embodiment, the subject suffers from a disorder selected from the group consisting of:

i) inflammatory diseases, such as Crohn's disease, irritable bowel syndrome (IBS), systemic lupus erythematous (SLE), nephritis;

ii) auto-immune diseases such as rheumatoid arthritis, ystemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis;

iii) cardiovascular diseases such as heart failure, kidney diseases (e.g. renal failure, nephritis, etc.) hypertension, pulmonary hypertension, cirrhosis, arteriosclerosis, pulmonary emphysema, pulmonary oedema; stroke, brain ischemia, myocardial impairment in sepsis;

iv) metabolic disease such as obesity, diabetes, anorexia, hyperphagia, polyphagia, hypercholesterolemia, hyperglyceridemia, hyperlipemia; various types of dementia such as senile dementia, cerebrovascular dementia, dementia due to genealogical denaturation degenerative diseases (e.g. Alzheimer's disease, Parkinson's disease, Pick's disease, Huntington's disease, etc.), dementia resulting from infectious diseases (e.g. delayed virus infections such as Creutzfeldt-Jakob disease), dementia associated with endocrine diseases, metabolic diseases, or poisoning (e.g. hypothyroidism, vitamin B12 deficiency, alcoholism, poisoning caused by various drugs, metals, or organic compounds), dementia caused by tumors (e.g. brain tumor), and dementia due to traumatic diseases (e.g. chronic subdural hematoma), depression, hyperactive child syndrome (microencephalopathy), disturbance of consciousness, anxiety disorder, schizophrenia, phobia;

muscles disorders such as skeletal muscle atrophy which is associated with bed rest, corticosteroid use, denervation, chronic renal failure, limb immobilization, neuromuscular disorders, sarcopenia of aging, and arthritis (WO2015035171); cancer, such as cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lympho epithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo- alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; muco epidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

As used herein, the term "blood sample" means any blood sample derived from the subject. Peripheral blood is preferred, and mononuclear cells (PBMCs) are the preferred cells. The term "PBMC" or "peripheral blood mononuclear cells" or "unfractionated PBMC", as used herein, refers to whole PBMC, i.e. to a population of white blood cells having a round nucleus, which has not been enriched for a given sub-population. Typically, these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis which will preferentially lyse red blood cells. Such procedures are known to the expert in the art.

As used herein, the term "predetermined reference value" refers to a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the expression level of the selected peptide in a group of reference, one can use algorithmic analysis for the statistic treatment of the expression levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUO0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

Method for identifying whether a subject was administered with an activator of the

ΡΡΑ β/δ pathway by calculating the ratio of γδ T cells and αβ T cells In a second aspect, the invention relates to a method for identifying whether a subject was administered with an activator of the PPARp/δ pathway comprising: i) quantifying the level of αβ T cells in a blood sample obtained from said subject; ii) quantifying the level of γδ T cells in a blood sample obtained from said subject; iii) calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii); iv) comparing the ratio determined at step iii) with a predetermined reference value, and v) concluding that the subject was administered with an activator of the PPARp/δ pathway when the ratio determined at step iii) is lower than the predetermined reference value or concluding that the subject was not administered with an activator of the PPARp/δ pathway when the ratio determined at step iv) is higher than the predetermined reference value.

As used herein, the term "αβ T cells" refers to T cells which express a T cell receptor (TCR) that is composed of a- and β-protein chains. The majority (>80%) of all CD3+ T cells in a normal, healthy person are αβ T cells. TCRs are associated with the invariant CD3 molecule that distinguishes T cells from B cells and all other types of immune cells. The majority of αβ T cells recognize the antigen in a so-called major histocompatibility complex (MHC) molecules-restricted fashion. There are two major classes of MHC molecules, MHC class I (MHC-I) and MHC class II (MHC-I), which trigger the TCRs of the two major subsets of αβ T cells, the CD8+ αβ T cells and CD4+ αβ T cells. The TCRs on CD4+ αβ T cells recognize MHC-II-peptide complexes whereas the TCRs on CD8+ αβ T cells recognize MHC-I-peptide complexes.

As used herein, the term "γδ T cells" refers to T cells which express a T cell receptor (TCR) that is composed of γ- and δ-protein chains. They are a distinct subset of CD3+ T cells featuring TCRs that are encoded by Vy- and νδ-gene segments (Morita et al, 2000; Carding and Egan, 2002). In humans, V01+-TCR chain expressing γδ T cells (V61+ T cells) predominate in epithelial or epithelia-associated/mucosal tissues of the skin, airways, digestive and urogenital tracts, and several internal organs, and constitute a minor fraction (<20%) of γδ T cells in peripheral blood. The TCRs of νδ1+ T cells recognize lipid antigens presented by MHC-related CD1 molecules. In human peripheral blood of healthy individuals γδ T cells make up 2-10% of total CD3+ T cells, and the majority (>80%) of peripheral blood γδ T cells are Vy2V02+-TCR chain-expressing γδ T cells (V 2V82 + γδ T cells) (Morita et al., 2000; Carding and Egan, 2002). The major subset of γδ T cells in human peripheral blood does not express CD4 or CD8 and its TCRs do not require MHC-restriction for antigen recognition.

In some embodiments, the αβ T cells or γδ T cells are quantified by cell sorting, more particularly by Fluorescence-activated cell sorting (FACS). Typically, cells are incubated with a fluorescently labelled antibody which recognizes a polypeptide present on the surface on the target cells population. The cells are then forced into a small nozzle of a cell sorter one at a time. They are then scanned by a fluorescence laser, separated according to their fluorescence and the target population can be collected. For example, the quantification of these cells is performed by contacting the blood sample with a binding partner (e.g antibody) for a cell marker of said cells. Typically, the quantification of these cells is performed by contacting the blood sample with several binding partners (e.g antibody) specific for following cell surface markers CD3, CD4, TCRc^ ΤΟΙγδ, CD44, CD62L, CD25, CD44 and CD8. These binding partners are coupled with fluorescent agents known in the art, such as fluorescein, isothiocyanate, phycoerythrin etc

Methods for determining whether a subject responds to a treatment with an activator of the ΡΡΑ β/δ pathway

The method according to the invention is suitable for determining whether a subject responds to a treatment with an activator of the PPARβ/δ pathway. More particularly, the method of the invention is suitable to determine whether a treatment with an activator of PPARβ/δ pathway increases the level of biomarkers such as Acaa2, Acadvl and Cptla or decrease the ratio of the level of αβ T cells to the level of γδ T cells quantified in a blood sample. Typically, firstly, the skilled man will measure the biomarker levels in blood sample before starting ΡΡΑΡνβ/δ pathway agonist treatment which corresponds to a reference value. Secondly, the skilled man will measure the biomarker levels in blood sample during the treatment. When the biomarkers level during treatment are increased compared to the biomarker level before treatment, it is indicated that the treatment was activating the ΡΡΑΡνβ/δ pathway and thus, the subject respond to this treatment.

Accordingly, in a third aspect, the invention relates to a method for determining whether a subject responds to a treatment with an activator of the ΡΡΑΡνβ/δ pathway comprising: i) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject before the treatment; ii) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject during the treatment; iii) comparing the expression determined at step ii) with a predetermined reference value, and iv) concluding that the subject responds to a treatment with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step ii) is higher than the predetermined reference value or concluding that the subject will not achieve a response to a treatment with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step ii) is lower than the predetermined reference value.

As used herein, the term "respond" refers to the response to a treatment of the subject suffering from a disorder. Typically such treatment induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. Accordingly, the survival time of the subject is increased with said treatment. In particular, in the context of the invention, the term "respond" refers to the ability of an activator of the ΡΡΑΡνβ/δ pathway to an improvement of the pathological symptoms, thus, the subject presents a clinical improvement compared to the subject who does not receive the treatment. The said subject is considered as a "responder" to the treatment. The term "not respond" refers to a subject who does not present any clinical improvement to the treatment with an activator of the ΡΡΑΡνβ/δ pathway treatment. This subject is considered as a "non-responder" to the treatment. Accordingly, the subject as considered "non-responder" has a particular monitoring in the therapeutic regimen.

As used herein, the term "subject" refers to a human. Particularly, the subject suffers from one of the disorders as described above. As used herein, the "predetermined reference value" refers to the level of biomarkers determined before the treatment with an activator of the ΡΡΑΡνβ/δ pathway.

In a fourth aspect, the present invention relates to a method for determining whether a subject responds to a treatment with an activator of the ΡΡΑΡνβ/δ pathway comprising: i) quantifying the level of αβ T cells in a blood sample obtained from said subject before the treatment; ii) quantifying the level of γδ T cells in a blood sample obtained from said subject before the treatment; iii) calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii); iv) calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii) during the treatment; v) comparing the ratio determined at step iv) with a predetermined reference value, and vi) concluding that the subject responds to the treatment with an activator of the ΡΡΑΡνβ/δ pathway when the ratio determined at step iv) is lower than the predetermined reference value or concluding that the subject does not respond to the treatment with an activator of the ΡΡΑΡνβ/δ pathway when the ratio determined at step iv) is higher than the predetermined reference value.

Methods and compositions for treating a subject

The method according to the invention is useful for a physician to monitor the subjects treated with an activator of the ΡΡΑΡνβ/δ pathway and to change the therapeutic regimen when the subject is determined as non-responder.

Accordingly, in a fifth aspect, the invention relates to a method of treating a subject in need thereof comprising the following steps: i) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject before the treatment; ii) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject during the treatment; iii) comparing the expression determined at step ii) with a predetermined reference value, iv) concluding that the subject will achieve a response to a treatment with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step ii) is higher than the predetermined reference value or concluding that the subject will not achieve a response to a treatment with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step ii) is lower than the predetermined reference value; and v) administering to the subject determined as non-responder, a therapeutically effective amount of a drug selected from the group consisting of an anti-inflammatory drugs, an anti-diabetic drugs, a chemotherapeutic agent, an immunotherapeutic agents or a radiotherapeutic agent. As used herein, the terms "treating" or "treatment" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the term "subject" refers to a human. Particularly, the subject suffers from one of the disorders as described above.

A "therapeutically effective amount" is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. As used herein, the "predetermined reference value" refers to the level of biomarkers determined before the treatment with an activator of the ΡΡΑΡνβ/δ pathway.

As used herein, the term "anti-inflammatory drugs" refers to a substance that reduces inflammation or swelling. The anti-inflammatory drugs are well known in the art and including, but not limited to non-steroidal anti-inflammatory drugs (e.g aspirin, ibuprofen, naproxen etc); aminosalicylates (e.g. azulfidine); corticosteroids etc

As used herein, the term "anti-diabetic drugs" refers to a substance used in diabetes to treat diabetes mellitus by lowering glucose levels in the blood. This kind of drugs are well known in the art and including, but not limited to Metformin, Thiazolidinediones, Exenatide; Liraglutide; Taspoglutide; Lixisenatide etc

As used herein, the term "chemotherapeutic agents" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Intl. Ed. Engl. 33 : 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and phannaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti- estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and phannaceutically acceptable salts, acids or derivatives of any of the above.

As used herein, the term "immunotherapeutic agents" refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Examples of common immunotherapeutic agents known in the art include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non- cytokine adjuvants. Alternatively the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells...). In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more checkpoint proteins. Checkpoint proteins regulate T-cell activation or function. Numerous checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD1 with its ligands PDL1 and PDL2 (Pardoll, Nature Reviews Cancer 12: 252-264, 2012). These proteins are responsible for co-stimulatory or inhibitory interactions of T-cell responses. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors include antibodies or are derived from antibodies. In some embodiments, the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-CTLA4 antibodies (e.g. Ipilimumab), anti-PDl antibodies (e.g. Nivolumab, Pembrolizumab), anti-PDLl antibodies, anti-TIM3 antibodies, anti-LAG3 antibodies, anti- B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies. Examples of anti-CTLA-4 antibodies are described in US Patent Nos: 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238. One anti-CTLA-4 antibody is tremelimumab, (ticilimumab, CP-675,206). In some embodiments, the anti- CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-D010) a fully human monoclonal IgG antibody that binds to CTLA-4. Another immune checkpoint protein is programmed cell death 1 (PD-1). Examples of PD-1 and PD-L1 blockers are described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos: WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699. In some embodiments, the PD-1 blockers include anti-PD-Ll antibodies. In certain other embodiments, the PD-1 blockers include anti-PD-1 antibodies and similar binding proteins such as nivolumab (MDX 1106, BMS 936558, ONO 4538), a fully human IgG4 antibody that binds to and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2; lambrolizumab (MK- 3475 or SCH 900475), a humanized monoclonal IgG4 antibody against PD-1; CT-011 a humanized antibody that binds PD-1 ; AMP-224 is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559 (MDX- 1105-01) for PD-L1 (B7-H1) blockade. Other immune- checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al, 2007, J. Immunol. 179:4202-4211). Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo et al, 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al, 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al, 2010, J. Exp. Med. 207:2187-94). In some embodiments, the immunotherapeutic treatment consists of an adoptive immunotherapy, as described by Nicholas P. Restifo, Mark E. Dudley and Steven A. Rosenberg ("Adoptive immunotherapy for cancer: harnessing the T cell response, Nature Reviews Immunology, Volume 12, April 2012). In adoptive immunotherapy, the patient's circulating lymphocytes, or tumor-infiltrated lymphocytes, are isolated in vitro, activated by lymphokines such as IL-2 and readministered (Rosenberg et al, 1988; 1989). The activated lymphocytes are most preferably be the patient's own cells that were earlier isolated from a blood sample and activated (or "expanded") in vitro.

As used herein, the term "radiotherapeutic agents" is intended to refer to any radio therapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation. For instance, the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy. Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy. In a sixth aspect, the invention relates to a method of treating a subject in need thereof comprising the following steps: i) quantifying the level of αβ T cells in a blood sample obtained from said subject before the treatment with an activator of the ΡΡΑΡνβ/δ pathway ; ii) quantifying the level of γδ T cells in a blood sample obtained from said subject before the treatment with an activator of the ΡΡΑΡνβ/δ pathway; iii) calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii); iv calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii) during the treatment; v) comparing the ratio determined at step iv) with a predetermined reference value; vi) concluding that the subject will achieve a response to the treatment with an activator of the ΡΡΑΡνβ/δ pathway when the ratio determined at step iv) is lower than the predetermined reference value or concluding that the subject will not achieve a response to the treatment with an activator of the ΡΡΑΡνβ/δ pathway when the ratio determined at step iv) is higher than the predetermined reference value; and vii) administering to the subject determined as non-responder, a therapeutically effective amount of a drug selected from the group consisting of an anti- inflammatory drugs, an anti-diabetic drugs, a chemotherapeutic agent, an immunotherapeutic agents or a radiotherapeutic agent.

Methods for identifying whether a high level athlete subject was administered with an activator of ΡΡΑ β/δ

The method according to the invention is suitable for identifying whether a high level athlete subject was administered with an activator of ΡΡΑΡνβ/δ. The World Anti-Doping Agency (WAD A) has classified activators of ΡΡΑΡνβ/δ to the prohibited list of substances. Typically, GW501516 was classified as a prohibited substance by WADA.

Accordingly, in a seventh aspect, the invention relates to a method for identifying whether an high level athlete subject was administered with an activator of ΡΡΑΡνβ/δ comprising: i) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject; ii) comparing the expression determined at step i) with a predetermined reference value, and iii) concluding that the subject was administered with an activator of ΡΡΑΡνβ/δ when the expression level of Acaa2, Acadvl and Cptla determined at step i) is higher than the predetermined reference value or concluding that the subject was not administered with an activator of ΡΡΑΡνβ/δ when the expression level of Acaa2, Acadvl and Cptla determined at step i) is lower than the predetermined reference value.

As used herein, the term "high level athlete subject" refers to rodent, a feline, a canine, an equidae and a primate. In a particular embodiment, the subject is a human. More particularly, a human who trains to compete in sports or exercises involving physical strength, speed, or endurance. The athlete has a natural aptitude for physical activities and participates to sport events (e.g national or international level competitions). In some embodiments, the high level athlete subject is a person who practices at least one sport selected from the group consisting of: race bike, run, karate, swimming, athletics (e.g competitive running, jumping, throwing or walking). In a particular embodiment, the high level athlete subject belongs to the family Equidae. In some embodiments, the high level athlete subject is a horse, typically, a horse which participates to horses racing.

In a eighth aspect, the invention relates to a method for identifying whether an high level athlete subject was administered with an activator of the ΡΡΑΡνβ/δ pathway comprising: i) quantifying the level of αβ T cells in a blood sample obtained from said high level athlete subject ; ii) quantifying the level of γδ T cells in a blood sample obtained from said high level athlete subject ; iii) calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii); iv) comparing the ratio determined at step iii) with a predetermined reference value, and v) concluding that the an high level athlete subject was administered with an activator of ΡΡΑΡνβ/δ pathway when the ratio determined at step iii) is lower than the predetermined reference value or concluding that the high level athlete subject was not administered with an activator of ΡΡΑΡνβ/δ pathway when the ratio determined at step iv) is higher than the predetermined reference value. The invention will be further illustrated by the following figures and examples.

However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: PPARji is functional in T cells and its activation adversely affects thymic T cell development. (A) Relative Acaa2, Acadvl, and Cptla mRNA levels in in vitro activated primary mouse CD4+ T cells treated for 48 hrs with 3 μΜ GW0742 (ΡΡΑΡνβ agonist) or 0.1% DMSO (vehicle). (B) Relative Cptla mRNA levels in thymus or lymph node tissue from mice treated for 48 hrs in vivo with GW0742 (0.3 mg/kg/day LP.) or vehicle (equivalent volume of DMSO). Data is normalized to DMSO control. (C) Total thymic cell counts from mice that received identical treatment as in (B). (D) Flow cytometric analysis of CD4 and CD8 expression on thymocytes from the same mice used in (C), treated with DMSO (vehicle) (left) or GW0742 (right). Relative percentages (mean ± s.e.m.) of DN (CD4-CD8-), DP (CD4+CD8+), SP4 (CD4+CD8-), and SP8 (CD4-CD8+) thymocytes are indicated. (E) Quantification of various thymocyte cell populations (horizontal axis) derived from data shown in (C) and (D). Data is pooled from 5 independent experiments (A) or from tissues obtained from 6 mice per treatment group (B-E), with flow plots shown in (D) being representative of latter treatment groups. Data shown in bar graphs (A-C,E) are expressed as mean ± s.e.m. *P<0.05 when compared to DMSO control (Mann- Whitney test).

Figure 2: Consequences of impaired T cell development in Tg T-PPARp mice for peripheral T cell populations. Quantification of various T cell populations in spleen (A) and lymph nodes (B).

Figure 3: Development of αβ T cells is impaired in Tg T-PPARp mice while γδ T cell production is unaffected. Data shown in bar graphs are expressed as mean ± s.e.m. *P<0.05 when compared to control (Mann- Whitney test).

Figure 4: Impaired T cell development observed in vivo in Tg T-PPARp mice can be reproduced in vitro in the OP9-DL1 co-culture model. (A) Expansion of control (open circles) vs Tg Τ-ΡΡΑΡνβ (closed circles) DN2/DN3 thymocytes after 3, 5, 7, and 12 days of co-culture with OP9-DL1 cells. Data are presented as number of cells per well (xl04), with 104 DN2/DN3 cells seeded on OP9-DL1 cells in a 24-well format at day 0. (B, C) Quantification of number of DN, DP, SP4 and SP8 cells (B) and number of DN1-4 cells (C) after one week of co-culture of DN2/DN3 thymocytes obtained from control and Tg T- ΡΡΑΡνβ mice. Data shown in graphs (B, C) are expressed as mean ± s.e.m. *P<0.05 when compared to control (Mann- Whitney test for (B-D).

Figure 5: Treatment with GW0742 increases Cptla mRNA levels in lymphoid tissues and blood. Mice were treated for a period of 6 weeks with the ΡΡΑΡνβ agonist GW0742 (3 mg/kg/day) supplemented in their food. Cptla mRNA levels in different lymphoid tissues and blood was measured by qPCR. *P<0.05, **P<0.01, ***P<0.001.

Figure 6: A single bout or regular exercise does not affect Cptla mRNA levels in lymphoid tissues and blood. Mice either underwent no exercise (sedentary control), a single bout of exercise on a treadmill (5° inclination, 35 cm/s during 1 hour), or 8 weeks of regular treadmill exercise (4 weeks adaptation with 3 exercises/week, followed by 3 weeks of 5 exercises/week, and 1 last week with 3 exercises). Cptla mRNA levels in different lymphoid tissues and blood was measured by qPCR. ND=not determined.

EXAMPLE:

Material & Methods

Animal models. Animals were maintained in a 12-h light, 12-h dark cycle and received food [UAR (Usine d'Alimentation Rationnelle), ViUemoisson sur Orge, France] and water ad libitum. All experimental procedures were conducted according to French legislation and approved by the Animal Care Committee of the Faculty of Medicine of the Nice Sophia Antipolis University (Comite Institutionnel d'Ethique Pour lAnimal de Laboratoire) (NCE/2013-116). Lck-Cre mice (Cre recombinase under control of the T cell specific Lck gene promoter were obtained from Jackson Laboratory (B6.Cg-Tg(Lck-cre)548Jxm/J, stock number 003802). CAG-Stop-ΡΡΑΡνβ mice, carrying a transgene containing the modified chicken β-actin promoter with the CMV/IE enhancer (CAG promoter) driving ΡΡΑΡνβ-IRES- Hygromycin chimeric mRNA expression under CRE-mediated recombination of a transcriptional Stop fragment, were described previously (11). Both strains are on the C57BL/6J background and were crossed to obtain double transgenic animals that overexpress ΡΡΑΡνβ specifically in T cells (Lck-Cre/CAG-Stop-ΡΡΑΡνβ mice). For convenience these double transgenic animals are referred to as Tg Τ-ΡΡΑΡνβ mice. It has previously been reported that Cre expression in Lck-Cre mice results in off-target effects including a decrease in thymic cellularity (toxic to CD4+CD8+ cells) (35). Therefore, the Lck-Cre mice were used as controls throughout this study. Wild type C57BL/6J mice obtained from Charles River (France) were used for in vitro and in vivo GW0742 treatment studies. For the latter in vivo studies, 15-week-old male C57B1/6J mice were injected (0.3 mg/kg/day LP.) with the ΡΡΑΚβ agonist GW0742 (Cayman Chemical) or vehicle (equivalent volume of DMSO) and tissues were harvested 48 hours later.

Primary CD4+ T cell isolation, activation, and culture. Spleen and lymph nodes

(cervical, brachial, mesenteric, and inguinal) were harvested from 10-week-old male wild- type C57BL/6J mice and converted into single cell suspensions in phosphate-buffered saline (PBS), pH 7.2, containing 0.5% fetal calf serum (FCS) and 2mM EDTA, using a gentleMACS Dissociator and appropriate gentleMACS C tubes by following the protocols provided by the manufacturer (Miltenyi Biotec). Single cell suspensions from both organs were pooled before continuing with the isolation of CD4+ cells by magnetic labeling and separation using CD4 (L3T4) microbeads and MACS Columns, respectively, following the protocols provided by the manufacturer (Miltenyi Biotec). The isolated CD4+ cells were cultured at a concentration of 4 x 105 cells/well in a 48-well plate in RPMI containing 10% FCS, 100 units/ml penicillin/streptomycin, and 50 μΜ 2-mercaptoethanol. Cells were treated with 3 μΜ GW0742 or 0.1% DMSO (vehicle) and activated with anti-CD3/anti-CD28 beads (Dynabeads mouse T-activator CD3/CD28, Invitrogen) following instructions provided by the manufacturer. After 48 hrs of treatment, the activated primary CD4+ cells were harvested and used directly for mRNA extraction and real-time quantitative PCR analysis. RNA extraction and quantitative real-time PCR. Total R A was extracted from cells or tissues with Triz 1 reagent following the supplier's protocol (Invitrogen). For standard quantitative real-time PCR reaction total RNA (1 μg) was reverse-transcribed using a QuantiTect Reverse Transcription Kit (Qiagen) on a Qcyclerll. Quantitative PCR was done using SYBR Premix Ex Taq (Tli RNase H Plus) (Ozyme) on a StepOne machine (Life Technologies). The mRNA levels of all genes reported were normalized to 36B4 transcript levels. Primer sequences are available upon request. For Mouse Fatty Acid Metabolism RT² Profiler™ PCR Arrays (SA Biosciences), total RNA (0.5 μg) was reversetranscribed using the RT2 First Strand reagents as part of the array kit per the manufacturer's instructions and qPCR was performed on a StepOne machine following detection protocols recommended by SA Biosciences. Data was analyzed using the excel spreadsheet provided online on the manufacturer's website.

Cell preparation and flow cytometry analysis. Thymi, spleens and lymph nodes (cervical, brachial, mesenteric, and inguinal lymph nodes pooled together) were harvested from control and Tg-T-PPARP mice and converted into single cell suspensions in phosphate-buffered saline (PBS), pH 7.2, containing 0.5% fetal calf serum (FCS) and 2mM EDTA, using a gentleMACS Dissociator and appropriate gentleMACS C tubes by following the protocols provided by the manufacturer (Miltenyi Biotec). Splenocyte single cell suspensions were subsequently depleted of red blood cells with RBC lysis buffer (Sigma). The resulting single cell suspensions were incubated with Fc Block (anti-mouse CD16/CD32 monoclonal antibody, BD Biosciences) for 15 min at 4°C before staining with fluorescently labeled primary antibodies for 20 min at 4°C in PBS, 0.5% BSA. CD3-fluorescein isothiocyanate, CD3- phycoerythrin, CD4-allophycocyanin, TCRP-phycoerythrin-Cy7, TCRy5- phycoerythrin, CD44-phycoerythrin-Cy7, CD62L-fluorescein isothiocyanate, CD25 -phycoerythrin, and CD44-phycoerythrin FACS antibodies were purchased from eBioscience. CD8-Peridinin chlorophyll antibody was from BD Biosciences. For blood cell stainings 100 μΐ whole blood (with EDTA) was directly incubate 1 : 1 with antibody solutions (2x concentrated) and incubated for 20 min at room temperature followed by addition of PBS, 0.5% FCS, 2 mM EDTA and centrifugation. The stained cell pellet was subsequently incubated with RBC lysis buffer and washed twice in PBS, 0.5%> BSA. For BrdU stainings, we used the BrdU staining kit (FITC) from eBioscience according to the manufacturers' instructions. Cells were gently washed twice and resuspended in PBS, 0.5%> BSA with DAPI. Stained cell preparations were analyzed using a BD FACSCanto II flow cytometer (BD Biosciences). Histochemistry. Thymi were fixed in 10% formalin before embedding in paraffin. Six- micrometer tissue sections were deparaffinized and then stained with hematoxylin and eosin (H&E) for histological analysis.

OP9-DL1 co-cultures The DN2/DN3 thymocytes used in co-cultures were isolated from thymocyte cell suspensions by labeling them using anti-CD25-phycoerythrin (PE) antibody and subsequently purifying them using an anti-PE multisort kit (Miltenyi Biotec). The resulting cell preparation still contained contaminating CD4+ cells, so the anti-PE microbeads were released from the cells using the MultiSort Release Reagent from the kit to allow for a second labeling with CD4 (L3T4) microbeads to deplete CD4+ cells. After CD4+ cell depletion, the purified DN2/DN3 cell preparations were cocultured on top of confluent monolayers of OP9-DL1 cells as previously described (15). DN2/DN3 thymocytes isolated from control and Tg T-PPARP mice were added at a concentration of 104 cells/well to 24- well plates that were seeded the day before with 104 OP9-DL1 cells in Opti-MEM medium supplemented with GlutaMAX (Gibco), 10% FCS, 100 units/ml penicillin/streptomycin, and 50 μΜ 2-mercaptoethanol. Interleukin 7 (R&D Systems) was added to the co-cultures at 2 ng/ml. After 3 days, half of the co-culture media was replaced by fresh media. At indicated times, the cocultures were incubated for 2 hrs with 10 μΜ BrdU before the cells were recovered and prepared for flow cytometry analysis as described above.

Statistics All calculations were performed using Prism 5.0 software (GraphPad, San Diego, CA). Statistical significance between groups was determined by the Mann- Whitney or Kruskal-Wallis nonparametric test as indicated.

Results

PPARp activation in vivo leads to a reduction in thymocyte numbers. To confirm that PPARP is functional in T cells, and that its activation induces genes implicated in fatty acid metabolism, we isolated primary naive CD4+ T cells from spleens of wild-type mice and cultured these cells in the presence of a PPARP agonist (3 μΜ GW0742) or vehicle (0.1 % DMSO) for 48 hrs. We subsequently quantified in these cells the relative mRNA levels of a set of 84 genes known to be implicated in fatty acid metabolism using a mouse fatty acid metabolism PCR array. Of these 84 genes, 3 were significantly increased more than 15%; Acetyl-CoA Acyltransferase 2 (Acaa2), very long-chain acyl-CoA dehydrogenase (Acadvl), and Cptla (Figure 1A). These three genes are known PPARP target genes and the enzymes they encode are rate-limiting factors in fatty acid oxidation. These results therefore demonstrate that PPARP is functional in primary mouse T cells and strongly suggest that, similar to muscle cells, ΡΡΑΡνβ activation in T cells leads to an increase in fatty acid oxidation. We also treated wild-type mice with the same ΡΡΑΡνβ agonist (0.3 mg/kg/day GW0742 LP.) or vehicle (equivalent volume of DMSO) for 48 hours before harvesting their thymus and lymph nodes and analysed Cptla mRNA levels in these tissues. Similar to the in vitro primary T cell cultures, this in vivo PPARP activation also led to an increase of mRNA levels of this PPARP target gene in these T cell-rich tissues (Figure IB). Surprisingly, we also observed during these in vivo studies that the 48 hr PPARP agonist treatment led to a 50% decrease in thymus cell numbers compared to vehicle-treated mice (Figure 1C). To study this effect in more detail, we analysed the different thymocyte populations based on their surface expression of CD4 and CD8 by flow cytometry in mice treated with either PPARP agonist or vehicle. As shown in Figure ID, the in vivo treatment with a PPARP agonist led to a small but significant reduction of the percentage of double positive (DP; CD4+CD8+) thymocytes compared to vehicle treatment (77.5±1.4% vs 81.5±0.6%). When presented as total cell numbers, these data demonstrate a 50% reduction in DP thymocytes in the agonist-treated mice compared to mice that received vehicle (Figure IE).

T cell specific overexpression of PPARp disrupts T cell development in the thymus. To investigate whether the thymic effects observed after systemic in vivo treatment with a PPARP agonist is a direct effect of PPARP action in developing T cells, we used a transgenic mouse model (Tg T-PPARP). This transgenic mouse model was developed to overexpress PPARP specifically in T cells in mice using a Cre-Lox system. For this we relied on the action of a Cre-recombinase, which's expression is driven by the lymphocyte protein tyrosine kinase (Lck) promoter that is active early on during T cell development, to remove a stop cassette that is flanked by LoxP sites, allowing transcription of the downstream PPARP transgene. Thymic size, weight and cell counts were reduced in these Tg T-PPARP mice compared to littermate control (Lck- Cre+/-) mice. Flow cytometry profiles, based on surface expression of CD4 and CD8 showed that, like with the in vivo PPARP agonist treatment, there was a significant reduction in the percentage of DP thymocytes in the Tg T-PPARP mice compared to littermate control mice (67.4±4.2% vs 79.4±2.1%). Furthermore, the percentage of double negative (DN; CD4-CD8-) thymocytes had significantly more than doubled in the Tg T-PPARP mice compared to littermate control mice (19.9±3.7% vs 7.5±1.4%). For cell numbers, this meant a significant 75% decrease in DP thymocytes with a trend, but no significant increase in the number of double negative (DN) thymocytes and a trend, but no significant decrease in mature CD4 or CD8 single-positive (SP) cells in the thymi of Tg T-PPARP vs control mice. Hematoxylin/Eosin staining of paraffin sections of thymi of control mice shows a clear corticomedullary differentiation, while this is lost in thymi of Tg T-PPARp mice.

Disrupted thymic T cell development in Tg T-PPARp mice results in a reduction in most, but not all, peripheral T cell populations

To investigate whether the disruption in T cell development in thymi from Tg T-

ΡΡΑΡνβ mice has consequences for T cell populations in peripheral lymphoid organs, we analysed the T cell populations in spleen, lymph nodes, and blood from Tg Τ-ΡΡΑΡνβ and littermate control mice. Total cell counts already show a reduction in total number of spleen and lymph node cells in these lymphoid organs when comparing Tg Τ-ΡΡΑΡνβ with littermate control mice. Flow cytometry data shows that the percentage of CD3+ cells is decreased significantly in all three tissues in the Tg Τ-ΡΡΑΡνβ mice compared to littermate control mice. Furthermore, when analysing CD4 and CD8 expression on these CD3+ cells, we observed a trend towards a decrease in percentages of both CD4+CD8- and CD4-CD8+ cells in most cases, accompanied by a significant 2.5- to 4-fold increase in the percentage of CD4-CD8- cells in all three lymphoid tissues examined from Tg Τ-ΡΡΑΡνβ mice compared to littermate control mice. When these results are presented as cell numbers for both spleen and lymph nodes, it becomes clear that disruption of T cell development in Tg Τ-ΡΡΑΡνβ mice results in a reduction (±60%) of the number of T lymphocytes (CD3+ cells), with more specifically a decrease in the number of CD4+CD8- (±70%) and CD4-CD8+ (±60%) cells, while CD4- CD8- cell numbers remain untouched in these peripheral lymphoid tissues (Figure 2A-B).

Disrupted thymic T cell development in Tg T-PPARp mice results in a reduction in αβ T cells, but does not affect γδ T cell production

CD4+ and CD8+ T cells largely consist of cells expressing the αβ T cell receptor (TCR), and cells expressing the γδ TCR, for the large majority, are neither expressing CD4 nor CD8. Therefore, we anticipated that the decrease in CD4+ and CD8+ cells in peripheral lymphoid tissues is illustrative of a decrease in αβ T cells, while the unchanged numbers of CD4-CD8- cells would suggest that γδ T cell production is not affected. We confirm that TCRβ+ cells in these lymphoid tissues indeed, for the large majority, consist of CD4+ and CD8+ cells, with only between 1 to 4% of cells being CD4-CD8-. In contrast, TCRy5+ cells from these lymphoid tissues mostly (±80%>) consist of CD4-CD8- cells. When analysing the TCRβ+ and TCRy5+ populations, as gated on CD3+ cells, in the different lymphoid tissues, a significant decrease in the percentage of TCRβ+ cells in Tg T-PPARβ compared to littermate control mice in all tissues is observed. On the other hand, the percentage of TCRy5+ cells is increased by 4- to 6-fold in these tissues when comparing Tg T-PPARβ to littermate control mice. However, when these results are presented as cell numbers, it becomes clear that this proportional increase of TCRy5+ cells is simply the consequence of a significant decrease (±70%) of the number of TCRP+ cells, and in fact the number of TCRy5+ cells in thymus, spleen, or lymph nodes from Tg T-PPARP mice doesn't differ from those in the same tissues from littermate control mice (Figure 3 A-C). Except for a slight but significant change in CD4+/CD8+ ratio in TCRP+ cells from blood from Tg T-PPARP mice compared to littermate control mice, no difference in subpopulation composition of lymphoid tissue TCRP+ or TCRy5+ cells is observed. We also analysed, based on expression levels of CD44 and CD62L, whether the reduction in CD4+ and CD8+ cells was accompanied by changes in the proportions of naive (CD44-CD62L+), memory (CD44+CD62L+), and effector (CD44+CD62L-) CD4+ or CD8+ T cell populations in peripheral lymphoid tissues. The percentage of naive T cells is consistently lower and the percentage of effector T cells is consistently higher in all three lymphoid tissues from Tg T-PPARP mice compared to littermate control mice, regardless whether they are CD4+ or CD8+. These differences don't reach statistical significance in blood CD4+ cells, most likely as a result of the large variation in values measured in blood samples compared to spleen and lymphnodes. When data is presented as cell numbers, we observe a significant reduction (81 to 94%) in the numbers of naive CD4+ and CD8+ T cells in both spleen and lymph nodes from Tg T-PPARP mice compared to littermate control mice. Numbers of memory and effector T cells also tend to be decreased but often the difference does not reach statistical significance.

Disruption of thymic T cell development in Tg T-PPARp mice occurs at DN4 stage of T cell development

To determine whether the decrease in DP thymocytes in thyrni from Tg T-PPARP compared to control mice is a consequence of upstream events, we analysed whether PPARP overexpression affected thymocyte populations that precede the DP cells. By using the surface markers CD25 and CD44 we distinguish the four different populations of DN thymocytes; DN1 (CD25-CD44+), DN2 (CD25+CD44+), DN3 (CD25+CD44-), and DN4 (CD25-CD44-). Flow cytometry analysis of these markers, gated on the DN population, demonstrate that there is a significant increase in the percentage of DN3 cells (35.2±2.6 vs 19.8±1.8%) in thymi from Tg T-PPARP mice compared to control mice. Furthermore, the percentage of DN4 cells is significantly decreased (40.0±2.8% vs 61.1±3.3%) in thymi from Tg T-PPARP mice compared to control mice. No significant change in percentage of DN1 or DN2 cells is observed. When data is presented as cell numbers, the only DN population that is significantly affected in thymi from Tg Τ-ΡΡΑΡνβ mice compared to control mice is DN4 (65% reduction).

The phenotype observed in Tg T-PPARp mice can be reproduced in vitro in the OP9-DL1 coculture model.

Next, we used the fact that DN2 and DN3 thymocytes express CD25 to isolate those populations from thymi from control and Tg Τ-ΡΡΑΡνβ mice by positive selection using magnetic beads. This approach resulted in the isolation of cell populations that were enriched in DN thymocytes (±90-95%) consisting mostly (90%) of a mix of DN2 (16-18%) and DN3 (73-75%o) thymocytes. However, since these cell preparations still contained a significant amount (3-9%) of SP4 thymocytes we performed a second phase of enrichment by depleting the cells of CD4+ cells, again using a magnetic bead approach. This resulted in a further enrichment/purification of the cells with the final cell preparation consisting for 99% of DN thymocytes that were almost entirely (98-99%) composed of DN2 (18-23%) and DN3 (75- 80%) thymocytes. These purified DN2/DN3 primary thymocyte preparations from both control and Tg Τ-ΡΡΑΡνβ mice were subsequently co-cultured with OP9-DL1 cells to allow us to follow their transition through the different stages of T cell development in vitro (15). First, we compared proliferative expansion of control versus Tg Τ-ΡΡΑΡνβ DN2/DN3 thymocytes after 3, 5, 7, and 12 days of co-culture with OP9-DL1 cells. After 12 days, the 104 DN2/DN3 cells/well that were originally seeded on OP9-DL1 cells in a 24-well format at day 0, had multiplied to reach 4.95±0.12 million cells/well in the case of control mice, but to significantly less (3.61±0.22 million cells/well) when the DN2/DN3 thymocytes were derived from Tg Τ-ΡΡΑΡνβ mice (Figure 4A). This significant difference in proliferative expansion of DN2/DN3 thymocytes derived from control compared to Tg Τ-ΡΡΑΡνβ mice already became apparent,after only 5 days of co-culture (Figure 4A). When we analysed the progeny of the DN2/DN3 thymocytes after 7 days of co-culture with OP9-DL1 cells, we observed that control DN2/DN3 progeny include a higher percentage of SP4 cells (43.5±1.0%) than progeny from Tg Τ-ΡΡΑΡνβ DN2/DN3 thymocytes (30.9±0.6%). Almost no SP8 cells had developed in these cocultures, as previously already observed under similar co-culture conditions (16, 17). The percentage of DP cells derived from the different DN2/DN3 thymocyte populations doesn't differ significantly. Furthermore, the percentage of cells still at the DN stage is higher in the progeny of DN2/DN3 thymocytes from Tg Τ-ΡΡΑΡνβ mice (51.1±1.0%) compared to control mice (37.3±0.7%). In fact, further analyses of the different DN populations revealed that only a low percentage of control DN2 thymocytes is left after 1 week co-culture (0.3±0.02%) compared to Tg Τ-ΡΡΑΡνβ DN2 thymocytes (1.9±0.2%). However, this difference does not reach statistical significance. The DN progeny of the control DN2/DN3 thymocytes mostly consists of newly transitioned DN4 thymocytes (58.2=1=3.1%), with still a significant percentage of DN3 thymocytes remaining (41.2=1=3.1%). In contrast, the majority of DN progeny derived from Tg Τ-ΡΡΑΡνβ DN2/DN3 thymocytes remains at the DN3 stage (74.2±1.4%), with only 23.5±1.6% of newly transitioned DN4 thymocytes. When these percentages were converted to actual cell numbers we observed that 1 week co-culture of Tg T-PPARp DN2/DN3 thymocytes with OP9-DL1 cells results in 50% less DN, 67% less DP, and 74% less SP4 cells compared to co-cultures performed with control DN2/DN3 thymocytes (Figure 4B). Furthermore, when we analysed in more detail the number of the different DN populations present after 1 week of co-culture, we observed that there are 80% less DN4 cells in the progeny from Tg T-PPARp DN2/DN3 thymocytes compared to their control counterpart (Figure 4C). No significant differences in cell numbers for the other DN subpopulations are observed.

Decreased proliferation and increased expression of genes implicated in fatty acid oxidation by Tg T-PPARp cells in in vitro OP9-DL1 co-culture model.

The results shown in Figure 3 A suggests that either there is less proliferation occurring in Tg TPPARP thymocytes co-cultured with OP9-DL cells, compared to control thymocytes, or there are more Tg T-PPARP cells dying. However, the latter possibility of increased cell death is unlikely since we did not observe more death cells in our Tg T-PPARP co-cultures, based on trypan blue stainings performed for the cell counts presented in Figure 4A, and the DAPI stainings performed for the FACS analysis (data not shown). Therefore, we analysed by BrdU stainings whether proliferation was affected in the thymocytes originating from the Tg T-PPARP mice compared to control mice. Due to the low number of DP, SP4, and SP8 cells present in the Tg T-PPARP cocultures (see Figure 4B), and the even lower number of BrdU positive cells in these populations, it is impossible to draw reliable conclusions regarding the proliferation rates in these cell populations. The same problem does not allow for an accurate determination of proliferation occurring in the remaining DN2 population (see Figure 4C). However, the degree of BrdU incorporation in the total cell population, the DN cells, and their DN3 and DN4 subpopulations could be determined. The results demonstrate that there is less cell proliferation occurring in the Tg T-PPARP co-cultures compared to control when looking at the total cell population (51.4±1.7% in control vs 32.4±1.4% in Tg T-PPARP). A similar difference in BrdU incorporation was observed in the DN population (45.2±4.2% in control vs 24.4±0.9% in Tg T-PPARP). Furthermore, while no significant difference in BrdU incorporation was observed in the DN3 population, the DN4 population originating from Tg T-PPARP DN2/DN3 thymocytes is proliferating significantly less than their control counterpart (49.9±2.8% in control vs 28.4±0.6% in Tg Τ-ΡΡΑΡνβ). As was mentioned in the introduction, successful development of T cells depends on a switch to glycolytic metabolism at the DN4 stage to allow for a proliferative burst of these cells (7, 8). As shown in Figure 1 A, activation of ΡΡΑΡνβ in mature T cells induces the expression of 3 enzymes that are rate- limiting factors for fatty acid oxidation, strongly suggesting that ΡΡΑΡνβ activation leads T cells to switch to lipid metabolism instead. We therefore investigated whether over-expression of ΡΡΑΡνβ in developing T cells has a similar effect. In the co-culture model, a significant difference in proliferative expansion of developing T cells became apparent after 5 days of co-culture (Figure 4A). Therefore, we decided to analyse whether an increase in the mRNA levels of genes encoding the aforementioned fatty acid oxidation enzymes precedes the observed decrease in proliferation at day 5. At day 4 of co-culture the mRNA levels for these 3 genes are indeed increased in developing T cells originating from Tg T-PPARβ compared to those that were derived from control mice. These results support a model where PPARβ activation/overexpression in developing T cells favours fatty acid- instead of glucose- oxidation in these cells, thereby hampering the proliferative burst normally occurring at the DN4 stage of T cell development, which negatively impacts all the subsequent populations of T cells that are derived from DN4 thymocytes.

Inventors have performed experiments on animal models, typically, mice were treated for a period of 6 weeks with the PPARβ agonist GW0742 (3 mg/kg/day) supplemented in their food. Treatment with GW0742 increases Cptla mRNA levels in lymphoid tissues and blood (Fig.5). Moreover, mice either underwent no exercise (sedentary control), a single bout of exercise on a treadmill (5° inclination, 35 cm/s during 1 hour), or 8 weeks of regular treadmill exercise (4 weeks adaptation with 3 exercises/week, followed by 3 weeks of 5 exercises/week, and 1 last week with 3 exercises). Inventors have demonstrated that a single bout or regular exercise does not affect Cptla mRNA levels in lymphoid tissues and blood (Fig.6).

Discussion

PPARβ has been shown to play a protective role in a growing list of inflammatory conditions (e.g. septic and non-septic shock, inflammatory bowel disease, and experimental autoimmune encephalomyelitis (EAE)), varying from acute to chronic inflammatory diseases and including several autoimmune diseases (9). Furthermore, PPARβ has been implicated in both the innate and adaptive immune system. In almost all the inflammatory disease models studied, PPARβ activation or overexpression leads to a decrease in inflammation, and deletion of PPARP leads to an aggravation of the inflammatory state. As a consequence, PPARP presents an interesting therapeutic target in a large variety of inflammatory conditions. Perhaps the novel role for PPARP in T cell development identified in this study might partially explain some of these previously reported anti-inflammatory effects of PPARP activation or overexpression. The thymic involution, followed by a decrease in peripheral β T cells, that are the consequences of overexpression/activation of PPARP in T cells, might be considered an additional mechanism by which PPARP can exert anti-inflammatory actions. So far, studies on the role of PPARP in immune cells have mostly focused on monocyte/macrophages (9). PPARP controls the inflammatory status of monocyte/macrophages in part by its association and disassociation with the transcriptional co-repressor B cell lymphoma-6 (BCL-6) protein (18). Another way PPARP can regulate inflammatory gene expression is by interacting with the p65 subunit of NFDB, a key transcriptional regulator of inflammation (19). Furthermore, two reports identified PPARP as a crucial signalling molecule controlling the phenotypic switch between pro -inflammatory Ml and anti-inflammatory M2 macrophages (20, 21). Except for these reports on macrophages, very little is known regarding the function of PPARD in other key inflammatory/immune cell types. Except for some reports demonstrating a role for PPARP in the T cell-mediated mouse EAE model (22-25), the study of the role of PPARP in T cells is still in its infancy. Most of the latter studies used global PPARP knockout mice or systemic agonist treatment and it is therefore difficult to define the contribution of PPARP absence/activation in T cells to the observed phenotypes. In that regard, our studies used mice that overexpress PPARP specifically in T cells, allowing us to determine the role of PPARP in T cell biology. Our observation that PPARP activation/overexpression increases the lipid oxidation potential of T cells confirms the role of PPARP as a switch for cellular fuel preference. Our study also suggests that stimulating lipid oxidation early on in T cell precursors will hamper their development into mature T cells. Furthermore, this only seems to affect οφ- but not γδ-Τ cell development, suggesting that development of οφ-Τ cells is more easily affected by manipulating their metabolism. Often, the level of expression is thought to be indicative of the significance of a protein's biological role in a certain tissue. However, this view might be somewhat simplistic since analysis of the expression pattern of PPARP has shown that in thymus the expression is one of the lowest of the tissues analyzed (26). Our results now demonstrate that this might be for good reason since a high level of expression would most likely severely affect T cell development. To our knowledge, this is the first study demonstrating a direct role for a member of the PPAR family in T cell development. One other group has previously reported that increased PPARy expression levels correlate with thymic involution and that treatment with PPARy agonist results in thymic involution (27, 28). However, the authors attributed these effects to an increase in PPARy-induced adipogenesis in thymic stroma that would indirectly compromise T cell development. While these authors did not study the direct role of PPARy in developing T cells, it cannot be excluded that their in vivo data with PPARy agonist treatment are (partially) the result of direct effects of PPARy in developing T cells. Further studies are needed to elucidate a potential direct role of PPARy in T cell development. Until recently, PPARy agonist were used widely for clinical purposes but despite their efficacy in diabetic patients, they displayed a variety of adverse effects, leading to their withdrawal or limitation of use in Europe and the US (29). As a result, focus is shifting to PPARP agonists as future candidates for therapeutic use. Although PPARP agonists are not yet in clinical use, human studies have been performed to test the efficacy of two compounds, GW501516 and MBX-8025, providing very encouraging findings for the treatment of metabolic disorders in dyslipidemic obese individuals (30-34). Although no adverse effects were reported in these human studies, further investigations with larger groups of individuals and longer period of treatment are required to fully establish the safety of these PPARP agonists. Furthermore, GW501516 is available from online retailers, often under the name of Endurobol, and the compound has been added since 2009 to the prohibited list of substances by the World Anti-Doping Agency (www.wada-ama.org).

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Claims

CLAIMS:

1. A method for identifying whether a subject was administered with an activator of the ΡΡΑΡνβ/δ pathway comprising: i) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject; ii) comparing the expression determined at step i) with a predetermined reference value, and iii) concluding that the subject was administered with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step i) is higher than the predetermined reference value or concluding that the subject was not administered with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step i) is lower than the predetermined reference value.

2. A method for identifying whether a subject was administered with an activator of the ΡΡΑΡνβ/δ pathway comprising: i) quantifying the level of αβ T cells in a blood sample obtained from said subject; ii) quantifying the level of γδ T cells in a blood sample obtained from said subject; iii) calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii); iv) comparing the ratio determined at step iii) with a predetermined reference value, and v) concluding that the subject was administered with an agonist of ΡΡΑΡνβ/δ when the ratio determined at step iii) is lower than the predetermined reference value or concluding that the subject was not administered with an agonist of ΡΡΑΡνβ/δ when the ratio determined at step iv) is higher than the predetermined reference value.

3. The method according to the claim 1 or 2, wherein, the activator of ΡΡΑΡνβ/δ is GW501516.

4. The method according to the claim 1 or 2, wherein, the activator of ΡΡΑΡνβ/δ is L- 165041.

5. The method according to the claim 1 or 2, wherein, the activator of ΡΡΑΡνβ/δ is a- lipoic acid.

6. The method according to the claim 1 or 2, wherein, the subject suffers from a disorder selected from the group consisting of: i) inflammatory diseases, such as Crohn's disease, irritable bowel syndrome (IBS), systemic lupus erythematous (SLE), nephritis; ii) auto-immune diseases such as rheumatoid arthritis, ystemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis; iii) cardiovascular diseases such as heart failure, kidney diseases (e.g. renal failure, nephritis, etc.) hypertension, pulmonary hypertension, cirrhosis, arteriosclerosis, pulmonary emphysema, pulmonary oedema; stroke, brain ischemia, myocardial impairment in sepsis; iv) metabolic disease such as obesity, diabetes, anorexia, hyperphagia, polyphagia, hypercholesterolemia, hyperglyceridemia, hyperlipemia; v) various types of dementia such as senile dementia, cerebrovascular dementia, dementia due to genealogical denaturation degenerative diseases (e.g. Alzheimer's disease, Parkinson's disease, Pick's disease, Huntington's disease, etc.), dementia resulting from infectious diseases (e.g. delayed virus infections such as Creutzfeldt-Jakob disease), dementia associated with endocrine diseases, metabolic diseases, or poisoning (e.g. hypothyroidism, vitamin B12 deficiency, alcoholism, poisoning caused by various drugs, metals, or organic compounds), dementia caused by tumors (e.g. brain tumor), and dementia due to traumatic diseases (e.g. chronic subdural hematoma), depression, hyperactive child syndrome (microencephalopathy), disturbance of consciousness, anxiety disorder, schizophrenia, phobia; vi) muscles disorders such as skeletal muscle atrophy which is associated with bed rest, corticosteroid use, denervation, chronic renal failure, limb immobilization, neuromuscular disorders, sarcopenia of aging, and arthritis; vii) cancer.

7. The method according to claim 2, wherein, αβ T cells or γδ T cells are quantified by cell sorting (FACS).

8. A method for determining whether a subject responds to a treatment with an activator of the ΡΡΑΡνβ/δ pathway comprising: i) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject before the treatment; ii) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject during the treatment; iii) comparing the expression determined at step ii) with a predetermined reference value, and iv) concluding that the subject responds to a treatment with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step ii) is higher than the predetermined reference value or concluding that the subject does not respond to a treatment with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step ii) is lower than the predetermined reference value.

9. A method for determining whether a subject responds to a treatment with an activator of the ΡΡΑΡνβ/δ pathway comprising: i) quantifying the level of αβ T cells in a blood sample obtained from said subject before the treatment; ii) quantifying the level of γδ T cells in a blood sample obtained from said subject before the treatment; iii) calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii); iv) calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii) during the treatment; v) comparing the ratio determined at step iv) with a predetermined reference value, and vi) concluding that the subject responds to the treatment with an activator of the

ΡΡΑΡνβ/δ pathway when the ratio determined at step iv) is lower than the predetermined reference value or concluding that the subject does not achieve respond to the treatment with an activator of the ΡΡΑΡνβ/δ pathway when the ratio determined at step iv) is higher than the predetermined reference value.

10. A method for identifying whether an high level athlete subject was administered with an activator of the ΡΡΑΡνβ/δ pathway comprising: i) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject; ii) comparing the expression determined at step i) with a predetermined reference value, and iii) concluding that the subject was administered with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step i) is higher than the predetermined reference value or concluding that the subject was not administered with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step i) is lower than the predetermined reference value.

11. A method for identifying whether an high level athlete subject was administered with an activator of the ΡΡΑΡνβ/δ pathway comprising: i) quantifying the level of αβ T cells in a blood sample obtained from said high level athlete subject ; ii) quantifying the level of γδ T cells in a blood sample obtained from said high level athlete subject ; iii) calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii); iv) comparing the ratio determined at step iii) with a predetermined reference value, and v) concluding that the an high level athlete subject was administered with an activator of ΡΡΑΡνβ/δ pathway when the ratio determined at step iii) is lower than the predetermined reference value or concluding that the high level athlete subject was not administered with an activator of ΡΡΑΡνβ/δ pathway when the ratio determined at step iv) is higher than the predetermined reference value.

12. The method according to claim 10 or 12, wherein, the high level athlete subject is a person who practices at least one sport selected from the group consisting of: race bike, run, karate, swimming, athletics (e.g competitive running, jumping, throwing or walking).

13. The method according to claim 10 or 11, wherein, the high level athlete subject is a horse.

14. A method of treating a subject in need thereof comprising the following steps: i) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject before the treatment; ii) quantifying the expression level of Acaa2, Acadvl and Cptla in a blood sample obtained from said subject during the treatment; iii) comparing the expression determined at step ii) with a predetermined reference value, iv) concluding that the subject responds to a treatment with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step ii) is higher than the predetermined reference value or concluding that the subject does not respond to a treatment with an activator of the ΡΡΑΡνβ/δ pathway when the expression level of Acaa2, Acadvl and Cptla determined at step ii) is lower than the predetermined reference value; and iv) administering to the subject determined as non-responder, a therapeutically effective amount of a drug selected from the group consisting of an anti-inflammatory drugs, an anti-diabetic drugs, a chemotherapeutic agent, an immunotherapeutic agents or a radiotherapeutic agent.

15. A method of treating a subject in need thereof comprising the following steps: i) quantifying the level of αβ T cells in a blood sample obtained from said subject before the treatment with an activator of the ΡΡΑΡνβ/δ pathway ; ii) quantifying the level of γδ T cells in a blood sample obtained from said subject before the treatment with an activator of the ΡΡΑΡνβ/δ pathway; iii) calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii); iv calculating the ratio of the level of αβ T cells quantified at step i) to the level of γδ T cells quantified at step ii) during the treatment; v) comparing the ratio determined at step iv) with a predetermined reference value; vi) concluding that the subject responds to the treatment with an activator of the ΡΡΑΡνβ/δ pathway when the ratio determined at step iv) is lower than the predetermined reference value or concluding that the subject does not respond to the treatment with an activator of the ΡΡΑΡνβ/δ pathway when the ratio determined at step iv) is higher than the predetermined reference value; and vii) administering to the subject determined as non-responder, a therapeutically effective amount of a drug selected from the group consisting of an anti-inflammatory drugs, an anti-diabetic drugs, a chemotherapeutic agent, an immunotherapeutic agents or a radio therapeutic agent.