Use of FOXK1 inhibitors for the treatment of inflammatory autoimmune diseases

Abstract

Targeting the ability of T cells to enhance aerobic glycolysis upon TCR stimulation or glucose supplementation represents an attractive strategy for the treatment of autoimmune diseases. Using a quantitative phosphoproteomic approach, the inventors have identified a new transcription factor called Foxk1 as being highly phosphorylated in T cells upon T Cell Receptor (TCR) engagement. The results also indicate that Foxk1 phosphorylation and nuclear translocation is dependent of the AKT-mTOR kinase activities. Using T-cell specific Foxk1 deficient mice (Foxk1- / -), the inventors demonstrated that Foxk1 is required for full T cell activation. Foxk1-deficient T cells exhibited reduced proliferation and cytokine secretion following TCR stimulation. Furthermore, T cells from Foxk1- / - mice were less prone to acquire an effector like phenotype than wild-type cells when challenged in vivo. Altogether, these results indicated that targeting can be envisioned to reduce auto-immune reactions. Thus, the present invention relates to the use of Foxk1 inhibitors for the treatment of inflammatory autoimmune diseases.

Description

[0001] USE OF FOXK1 INHIBITORS FOR THE TREATMENT OF INFLAMMATORY AUTOIMMUNE DISEASES

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular immunology.

[0004] BACKGROUND OF THE INVENTION:

[0005] T cells are a type of white blood cell that play a crucial role in the adaptive immune system. They can recognize and eliminate foreign antigens, such as bacteria, viruses, or cancer cells, by binding to them with their T cell receptors (TCRs). T cells can also regulate the activity of other immune cells, such as B cells or macrophages, by releasing cytokines or expressing costimulatory molecules. However, in some cases, T cells can also react against self-antigens, which are normally tolerated by the immune system. This can lead to autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis, multiple sclerosis, or psoriasis. Autoimmune diseases are characterized by chronic inflammation and tissue damage caused by the aberrant activation of T cells. The functions of T cells are intimately linked to metabolic programs. During immune responses, T cells indeed undergo a metabolic reprogramming characterized by increased aerobic glycolysis, nutrient uptake and macromolecules synthesis. These processes are crucial for cell growth, differentiation, and acquisition of effector functions. In this regard, the AKT-mTOR signalling pathway has been shown to be an essential hub for regulating T cell metabolism and, consequently cell fate. Accordingly, impairing the ability of T cells to enhance aerobic glycolysis upon TCR stimulation or glucose supplementation represents an attractive strategy for the treatment of autoimmune diseases.

[0006] Among the metabolic regulators of T cell function, the forkhead box (Fox) family of transcription factors has emerged as a key player. Fox proteins modulate the expression of genes involved in cell cycle, apoptosis, differentiation, and metabolism. In particular, FoxOl and FoxO3 have been extensively studied in T cells and shown to control their quiescence, activation, and memory formation. However, less is known about other members of the Fox family, such as Foxkl, which belongs to the subfamily K of Fox proteins. Foxkl is highly expressed in skeletal muscle and regulates its growth and regeneration. Recently, it has been reported that FoxKl is also expressed in lymphocytes and could plays a role in their functions Sukonina, Valentina, et al. "FOXK1 and FOXK2 regulate aerobic glycolysis. " Nature 566. 7743 (2019): 279-283).

[0007] SUMMARY OF THE INVENTION:

[0008] The present invention is defined by the claims. In particular, the present invention relates to the use of Foxkl inhibitors for the treatment of inflammatory autoimmune diseases.

[0009] DETAILED DESCRIPTION OF THE INVENTION:

[0010] The present invention relates to a method of treating an inflammatory autoimmune diseases in patient in need thereof comprising administering to the patient a therapeutically effective amount of a Foxkl inhibitor.

[0011] As used herein, the term “patient” is interchangeable with the term “individual” or “subject”, and may refer to a subject to be treated by the methods disclosed herein. In particular, the patient suffers from an inflammatory autoimmune disease. In some embodiments, the patient is an infant. In some embodiments, the patient is a child. In some embodiments, the patient is an adult.

[0012] As used herein, the term “inflammatory autoimmune disease” refers to is a disease that is mediated by T cells, more particularly autoreactive T cells.

[0013] As used herein, the term “T cell” has its general meaning in the art and represent an important component of the immune system that plays a central role in cell-mediated immunity. T cells are known as conventional lymphocytes as they recognize the antigen with their TCR (T cell receptor for the antigen) with presentation or restriction by molecules of the complex major histocompatibility. There are several subsets of T cells each having a distinct function such as CD8+ T cells, CD4+ T cells, and gamma delta T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. As used herein, the term “autoreactive T cell” refers to a T cell is specific for an autoantigen. As used herein, the term “autoantigen” is used to refer to antigens produced by an individual that are recognized by the immune system of that individual. In some embodiments, an autoantigen is associated with an autoimmune disease. In general, an autoantigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, etc. In some embodiments, an autoantigen is or comprises a polypeptide. Those of skill in the art are familiar with a variety of agents, including polypeptides, that can act as autoantigens, and particular that are recognized in immune reactions associated with autoimmunity diseases, disorders and / or conditions.

[0014] In some embodiments, the autoimmune inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic scleroderma, sclerosis, systemic sclerosis, multiple sclerosis (MS), spino-optical MS, primary progressive MS (PPMS), relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and / or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus (including nephritis, cerebritis, pediatric, non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki's disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AH4A), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus, optionally pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis); subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis, optionally benign monoclonal gammopathy or monoclonal garnmopathy of undetermined significance, MGUS, peripheral neuropathy, paraneoplastic syndrome, channel opathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channel opathies of the CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressier's syndrome, alopecia greata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampler's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthamopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemiareperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman- Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis (e.g. chronic pancreatitis), polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired splenic atrophy, infertility due to antispermatozoan antibodies, non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as Leishmania, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, ischemic re-perfusion disorder, reduction in blood pressure response, vascular dysfunction, antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, reperfusion injury of myocardial or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic intractable inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer, valvulitis, endometriosis and graft-versus-host-disease (GVHD).

[0015] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients 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 patient 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 patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0016] In particular, the Foxkl inhibitor of the present invention is particularly suitable for reducing proliferation and cytokine secretion of T cells, in particular autoreactive T cells, following TCR stimulation.

[0017] As used herein, the term "Foxkl" refers to a member of the forkhead family of transcription factors and refers to the Forkhead Box Protein KI that is encoded by the Foxkl gene (Gene 221937). Foxkl is also known as Mnf. Foxkl induces aerobic glycolysis by upregulating the enzymatic machinery required for this (for example, hexokinase-2, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase), while at the same time suppressing further oxidation of pyruvate in the mitochondria by increasing the activity of pyruvate dehydrogenase kinases 1 and 4. Together with suppression of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 this leads to increased phosphorylation of the El a regulatory subunit of the pyruvate dehydrogenase complex, which in turn inhibits further oxidation of pyruvate in the mitochondria — instead, pyruvate is reduced to lactate. Suppression of Foxkl induces the opposite phenotype. An exemplary amino acid sequence of Foxkl is shown as SEQ ID NO:1.

[0018] SEQ ID NO : 1 >sp | P85037 | FOXK1_HUMAN Forkhead box protein KI OS=Homo sapiens OX=9606 GN=FOXK1 PE=1 SV=1 MAEVGEDSGARALLALRSAPCSPVLCAAAAAAAFPAAAPPPAPAQPQPPPGPPPPPPPPL PPGAIAGAGSSGGSSGVSGDSAVAGAAPALVAAAAASVRQSPGPALARLEGREFEFLMRQ PSVTIGRNSSQGSVDLSMGLSSFI SRRHLQLSFQEPHFYLRCLGKNGVFVDGAFQRRGAP ALQLPKQCTFRFPSTAIKIQFTSLYHKEEAPASPLRPLYPQI SPLKIHI PEPDLRSMVSP VPSPTGTI SVPNSCPASPRGAGSSSYRFVQNVTSDLQLAAEFAAKAASEQQADTSGGDSP KDESKPPFSYAQLIVQAI SSAQDRQLTLSGIYAHITKHYPYYRTADKGWQNSIRHNLSLN RYFIKVPRSQEEPGKGSFWRIDPASEAKLVEQAFRKRRQRGVSCFRTPFGPLSSRSAPAS PTHPGLMSPRSGGLQTPECLSREGSPI PHDPEFGSKLASVPEYRYSQSAPGSPVSAQPVI MAVP P RP S S L VAK P VAYMP AS I VT S QQ P AGHAI H WQQAP T VTMVRWT T SAN SAN GY I L TSQGAAGGSHDAAGAAVLDLGSEARGLEEKPTIAFATI PAAGGVIQTVASQMAPGVPGHT VTILQPATPVTLGQHHLPVRAVTQNGKHAVPTNSLAGNAYALTSPLQLLATQASSSAPW VT RVC E VG P KE P AAAVAAT AT T T PAT AT TASASASSTGEP E VKRS RVE EPS GAVT T P AGV IAAAGPQGPGTGE

[0019] As used herein, the term “Foxkl inhibitor” refers to any compound natural or not which is capable of inhibiting the activity or expression of Foxkl. The term encompasses any Foxkl inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition or downregulation of a biological activity associated with activation of the Foxkl. The term also encompasses inhibitor of expression. In some embodiments, the Foxkl inhibitor is selective over the other member of the forkhead family of transcription factors, especially Foxk2. By “selective” it is meant that the inhibition of the selected compound is at least 10-fold, preferably 25-fold, more preferably 100-fold, and still preferably 300-fold higher than the inhibition of the other NME kinases. The Foxkl inhibition of the compounds may be determined using various methods well known in the art.

[0020] In some embodiments, the Foxkl inhibitor is a small organic molecule.

[0021] In some embodiments, the Foxkl inhibitor is an inhibitor of Foxkl expression. An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In some embodiments, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of Foxkl mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of Foxkl, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding Foxkl can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. The Foxkl gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that Foxkl gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing Foxkl. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0022] According to the invention, the Foxkl inhibitor is administered to the patient in a therapeutically effective amount.

[0023] As used herein, the expression "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions 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 with the active ingredients; 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, typically 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. Typically the active ingredient of the present invention (e.g. Foxkl inhibitor) is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0024] A further aspect of the invention relates to a method for screening a plurality of test substances useful for the treatment of an inflammatory autoimmune disease in a patient in need thereof comprising the steps consisting of (a) testing each of the test substances for its ability to inhibit the activity or expression of Foxkl and (b) and positively selecting the test substances capable of said inhibition.

[0025] The term “test substance” refers generally to a material that is expected to decrease, reduce, suppress or inhibit the kinase activity or expression of Foxkl, which include small molecules, high molecular weight molecules, mixture of compounds such as natural extracts or cell or tissue culture products, biological material such as proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamer, RNAzymes and DNAzymes, or glucose and lipids, but is not limited thereto. These materials are obtained from synthetic or natural compound libraries and the methods to obtain or construct libraries are known in the art. For example, synthetic chemical library may be obtained from Maybridge Chemical Co. (UK), Comgenex(USA), Brandon Asociates(USA), Microsource(USA) and Sigma-Aldrich(USA). The chemical library of natural origin may be obtained from Pan Laboratories (USA) and MycoSearch(USA). Further test substances may be obtained by various combinatorial library construction methods known in the art including for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries. Test substance of a library may be composed of peptides, peptoides, circular or liner oligomeric compounds, template based compounds such as benzodiazepine, hydantoin, biaryls, carbocyclic and polycyclic compounds such as naphthalene, phenothiazine, acridine, steroids and the like, carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocyclic compounds such as triazine, indole, thiazolidine and the like, but does not limited thereto.

[0026] 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.

[0027] FIGURES:

[0028] Figure 1: (A) Western blot of protein lysates of purified CD4+T cells from Foxklfl / fl(wildtype) and Foxkl’7’ (knockout) mice. (B) Similar cells were stimulated for 48h (B) or 72h (C) with anti-CD3 plus anti-CD28 antibodies to assess cytokine secretion (B) and proliferation (C). (D) Purified CD8+ T cells from OT-I Foxklfl / fl and Foxkl- / - mice were stimulated for 72 h with antigen presenting cells pulsed with OVA peptides (T4; 10-8M or N4; 10-10M), PI or IL- 7 and were analyzed for IFN-g secretion.

[0029] Figure 2: Analysis of Foxkl deficient T cells. (A,B) Purified CTV-labelled naive CD4+ T cells from Foxklfl / fl or Foxkl- / - mice, were injected into CD3s- / - mice. After 6 days, spleens and lymph nodes were harvested and CD4+ T cells were analyzed by flow cytometry for CTV dilution and CD62L and CD44 expression. (C,D) Purified naive CD4+ T cells from Foxklfl / fl or Foxkl- / - mice were injected into CD3s- / - mice. Spleens and lymph nodes were harvested 21 days later and CD4+ T cells were analyzed by flow cytometry for CD62L, CD44, KLRG1 and CD127 expression. Naive, Central Memory (CM) and Effector memory (EM) cells were identified using CD62L and CD44 markers. Short Live Effector Cells (SLECs) and Memory Precursor Effector Cells (MPECs) were identified as KLRGlhighCD1271ow and KLRGllowCD127high cells respectively. Data are representative of two or three independent experiments and are presented as the mean ± SD (3-4 mice / group). The statistical analysis was performed using Welch’s t-tests *p < 0.05, **p < 0.01, ***p < 0.001.

[0030] Figure 3: Impact of Foxkl inactivation on T cell metabolic processes. (A) Purified CD4+ T cells from Foxklfl / fl and Foxkl- / - mice were left untreated (non-stimulated; NS) or were stimulated for 24h with anti- CD3s plus anti-CD28 antibodies or with phorbol myristate plus ionomycin (PI) or maintained quiescent with IL-7, and analyzed for CD71 and CD98 expression by flow cytometry. PI was used as positive control and IL-7 was used as negative control. (B) Analysis of oxygen consumption rate (OCR) on addition of oligomycin (Oligo), fluorocarbonyl cyanide phenylhydrazone (FCCP) and rotenone plus antimycin A (Rot / AA) in CD4+T cells from Foxklfl / fl and Foxkl- / - mice. OCR at maximal respiration is shown on the right. (C,D) Analysis of extracellular acidification rate (ECAR) on addition of glucose (GLC), oligomycin (Oligo) and 2-deoxyglucose (2-DG) C or following CD3 D / CD28 stimulation D in CD4+T cells from Foxklfl / fl and Foxkl- / - mice. (E) Glucose uptake of purified CD4+ T cells from Foxklfl / fl and Foxkl- / - mice, left untreated (non-stimulated; NS) or stimulated for 48h with anti-CD3 antibody in absence or presence of anti-CD28 antibody, or with IL-7. (F) Lactate secretion of similar cells as in (g). Data are representative of two independent experiments and are presented as the mean ± SD (3-4 mice / group). The statistical analysis was performed using Welch’s t-tests *p < 0.05, **p < 0.01, ***p < 0.001.

[0031] Figure 4: Identification of Foxkl targeted genes in T cells. qChIP analysis of Foxkl at the promoters of autophagy genes of unstimulated (Oh) and 4h stimulated CD4+ T cells.

[0032] EXAMPLE:

[0033] EXAMPLE 1

[0034] T lymphocytes play a key role in the immune response and their functions are intimately linked to metabolic programs. During immune responses, T cells undergo a metabolic reprogramming characterized by increased aerobic glycolysis, nutrient uptake and macromolecules synthesis. These processes are crucial for cell growth, differentiation, and acquisition of effector functions. In this regard, the AKT-mTOR signalling pathway has been shown to be an essential hub for regulating T cell metabolism and, consequently cell fate.

[0035] Using a quantitative phosphoproteomic approach, we have identified a new transcription factor called Foxkl as being highly phosphorylated in T cells upon T Cell Receptor (TCR) engagement. Our results also indicate that Foxkl phosphorylation and nuclear translocation is dependent of the AKTmTOR kinase activities. Using T-cell specific Foxkl deficient mice (Foxkl- / -), we demonstrated that Foxkl is required for full T cell activation. Foxkl -deficient T cells exhibited reduced proliferation and cytokine secretion following TCR stimulation. Furthermore, T cells from Foxkl- / - mice were less prone to acquire an effector like phenotype than wild-type cells when challenged in vivo.

[0036] Global proteomic analysis of TCR-stimulated CD4+ and CD8+ T cells from Foxkl deficient mice revealed defective expression of critical proteins involved in metabolism, and in particular of effectors and enzymes of the glycolysis pathway. Accordingly, Foxkl deficient T cells had reduced maximal mitochondrial respiratory capacity compared to wild-type and exhibited an impaired ability to enhance aerobic glycolysis upon TCR stimulation or glucose supplementation.

[0037] Altogether, these results indicated that Foxkl is a major regulator of T cell metabolism and thus, of T cell effector functions. Its molecular targeting can be envisioned to reduce autoimmune reactions.

[0038] EXAMPLE 2

[0039] Results

[0040] TCR stimulation triggers Foxkl nuclear translocation in an Akt-mTOR dependent manner

[0041] Foxkl and Foxk2 are present in all mouse tissues, mRNA expression databases indicate high expression of Foxkl in T lymphocytes (biogps.org). Our previous quantitative phosphoproteomic analysis of primary CD4+ and CD8+ T cells also identified Foxkl but not Foxk2 as phosphorylated at multiple serine (Ser) and threonine (Thr) residues following TCR engagement12, 13. In addition, immunoblot analysis of cellular extracts from mouse lymphoid organs showed low expression of Foxkl in the thymus, but a substantial abundance of Foxkl proteins in purified mature CD4+ and CD8+ T cells, while Foxk2 was undetectable in these conditions (Data not shown). Protein abundance of Foxkl was also higher in naive T cells compared with activated T cells (Data not shown). Analysis of the cellular localization of Foxkl in resting CD4+ and OT-I CD8+ T cells showed detection of the TF mainly in the cytoplasm (Data not shown). Of interest, TCR stimulation triggers nuclear accumulation of Foxkl, starting after 2 min and peaking at 10 min. To better characterize the upstream signalling pathways promoting Foxkl nuclear translocation, we treated T cells with specific inhibitors of mTOR (Ku63794), Aktl / 2 (AKTi VIII), or Gsk3 (ARA014418) and reassessed the transcription factor’s cellular localization. Akt or mTOR inhibition fully prevented Foxkl nuclear translocation following TCR stimulation. In contrast, Gsk3 inhibition enhanced Foxkl accumulation in the nucleus even in the absence of TCR engagement (Data not shown). Therefore, these results indicated that Foxkl is mainly localized in cytoplasm in resting T cells and translocates into the nucleus after TCR stimulation in an Akt-mTOR signaling pathwaydependent manner.

[0042] Foxkl is required for full activation of T cells

[0043] To evaluate the role of Foxkl in T cells, we crossed the Foxklfl / flmouse11with a CD4-Cre transgenic mouse in order to obtain mice whose T cells lack Foxkl proteins (hereafter Foxkl- / -) (Data not shown). Analysis of thymi from Foxkl- / - mice showed that their T cells developed normally, with similar cell numbers (Data not shown). The periphery was populated with T cells with a normal phenotype, although we observed a slight reduction of the numbers of CD4+ T cells, CD8+ T cells and Treg cells in spleens and lymph nodes (Data not shown). To evaluate the importance of Foxkl during T cell activation, we purified CD4+ T cells from Foxkl- / - and control mice and analysed their phenotypes after TCR stimulation with anti-CD3s in the presence or absence of anti-CD28. Foxkl -deficient cells showed a normal upregulation of the activation markers CD69 and CD44, but much less proliferation and secretion of IL-2 and IFN- y as compared with controls (Fig. ID). In addition, when naive T cells were cultured in vitro under skewing conditions promoting T helper (Th) cell differentiation, less IFN-g+ and IL-4+ producing cells were detected in Foxkl- / - T cells than in control cells under Thl- and Th2- polarizing conditions respectively (Data not shown). In contrast, no major differences were observed under Treg and Thl7 skewing conditions.

[0044] To examine the functions of Foxkl in CD8+ T cells, Foxkl- / - mice were crossed with OT-I transgenic mice expressing a TCR specific for the ovalbumin (OVA). Purified OT-I CD8+ T cells were co-incubated with irradiated splenocytes from CD3s- / - mice (acting as antigen- presenting cells), pulsed with increasing doses of the ovalbumin-derived peptide (N4), or its variant of lower affinity (T4). When stimulated with N4 or T4 peptides, the absence of Foxkl resulted in much less production of IFN-y and reduced proliferation of OT-I CD8+ T cells compared with control cells, despite their similar capacity to integrate certain signals, as reflected by increased CD69 and CD44 expression levels compared with the unstimulated condition (Fig. 2A).

[0045] To evaluate the proliferative capacity of Foxkl -deficient T cells in vivo, naive CD4+ T cells were labelled with Cell Trace Violet (CTV) and injected into CD3s- / - mice, which are devoid of T cells. Because of the lymphopenic environment of the hosts, injected T cells proliferate in a manner dependent on TCR-self antigen interactions14, 15. Flow cytometry analysis after 6 days showed that a lower proportion of Foxkl -deficient T cells had completely diluted the CTV, indicative of slower rates of proliferation (Fig. 2B). Moreover, staining of injected cells with CD62L and CD44 markers, revealed reduced ability of Foxkl- / - T cells to differentiate into effector memory T (Tern) cells (Fig. 2C). To explore this defect further, we performed a similar analysis 21 days following injection. Although Foxkl -deficient T cells eventually succeed in differentiating into Tern cells, the relative proportions of naive cells, central memory cells (Tcm) and Tern cells indicate that they lag behind control cells in their ability to differentiate into effector / memory cell populations (Fig. 2D). In addition, analysis of memory phenotypes showed that transferred Foxkl- / - T cells were more prone to differentiate into KLRGllowCD127hlghmemory precursor effector cells (MPECs) than into KLRGllhlghCD127lowshort-lived effector cells (SLECs), compared with their control counterparts (Fig. 2E). Consistently, we also found increased percentages of the PD-llowCD27hlghpopulation with injected Foxkl -deficient T cells, indicative of the persistence of low-activated cells (Data not shown). Taken together, these results demonstrate that Foxkl is required to trigger T cell activation and regulate effector T cell differentiation.

[0046] Foxkl regulates metabolic pathways of T cells

[0047] To investigate the molecular mechanisms by which Foxkl regulates T cell activation, we performed transcriptomic analysis on purified naive CD4+ T cells from Foxkl- / - and control mice that were left resting or activated with anti-CD3 plus anti-CD28 for 6 and 24 hours. The absence of Foxkl during T activation led to a decreased expression of 355 genes and an increased expression of 618 other genes (Data not shown). Gene set enrichment analysis (GSEA) using Hallmark gene sets indicated that the absence of Foxkl decreased the expression of target genes of c-Myc and E2F, of genes involved in the Akt-mTOR signaling pathway, and in processes of oxidative phosphorylation (OXPHOS) and glycolysis (Data not shown). For the latter, transcripts with reduced expression included the glucose receptor (Slc2al) and glycolytic enzymes (Hk2, Pfkl, Pgkl, Pgaml, Tpil, Ldhd) (Data not shown). Given the importance of metabolic changes on cellular function and T-cell differentiation, we then performed a GSEA analysis with immune-specific gene signatures. This analysis indicated that activated Foxkl- deficient CD4+ T cells failed to switch to an effector / memory cell state as evidenced by their inability to upregulate Tbx21. Irf8. Ifng, 112, Pomes, while remaining positive for some naive markers such as Ccr7 and Bach2 (Data not shown). As observed previously in Figure 2, we noted that this differentiation defect is partial, since the expression of some markers characterizing the transition of naive to effector / memory cells (Cd44, Sell, Il7f) is regulated similarly to that of controls. (Data not shown).

[0048] To further characterize the molecular pathways controlled by Foxkl, we determined by quantitative mass spectrometry (MS) analysis, the proteomes of control and Foxkl- / - CD4+ T cells activated with CD3 / CD28 for 24 and 48 hours. After normalization and filtering, more than 8000 proteins were identified and quantified across all conditions. Cell stimulation of Foxkl- / - CD4+ T cells resulted in a change in the abundance of 1376 proteins compared with control cells, with no major impact on the increase in total protein mass associated with cell activation (Data not shown). GSEA with Hallmark terms on proteomic data showed enrichment of similar terms with those identified by transcriptomic analysis (Data not shown). Thus, gene signatures associated with Myc targets and mTor signaling were significantly repressed in Foxkl -deficient cells. Although induction of Myc expression itself was unaffected, Foxkl- deficient T cells exhibits reduced expression of some of its targets such as the heterodimer SLC7A5 / SLC3A2(also CD98) and the Transferrin receptor TFRC (also CD71) required for the uptake of essential amino-acids and iron, respectively (Fig 3A). Concomitant with transcriptomic data, Foxkl is also required to induce the high-level protein abundance of the glucose receptor SLC2A1 and crucial components of glycolysis pathways (HK2, PFKL, ALDOA, PGK1, PGAM1, SLC16A3) that is reached in control cells following TCR stimulation (Data not shown).

[0049] In order to functionally assess the metabolic processes affected by protein expression changes, we analyzed aerobic glycolysis and OXPHOS of TCR-activated CD4+ T cells. We observed that Oxygen consumption rate (OCR) at maximal capacity and extracellular acidification rate (ECAR) in response to glucose and to CD3 D / CD28 stimulation were significantly decreased in Foxkl- / - T cells compared with their control counterparts (Fig. 3B-D). Consistently, in the absence of Foxkl, TCR stimulation failed to increase glucose uptake and production of lactate to levels comparable to those in control T cells (Fig. 3E & 3F). Overall, these analyses revealed that Foxkl is required to induce mTor-mediated metabolic processes enabling effector T cell differentiation.

[0050] Foxkl represses autophagy transcriptional programs

[0051] Analysis of transcriptomic data from the first 6h of cell activation showed a predominant increase in transcripts in Foxkl- / - T cells compared with controls, which is not observed at 24h (Data not shown). This suggested that Foxkl could exert a direct repressive function on the transcription of certain genes shortly after TCR stimulation. In myoblasts, Foxkl has been shown to repress autophagy and atrophy genes9. At 6h following activation, GSEA analysis revealed a significant enrichment for the KEGG autophagy pathway driven by core components of the autophagy machinery such as Autophagy-related protein family members (AtglOl, Atgl2, Atgl3, Atgl4, Alg2a), Wdr45, Ulkl, Taxlbpl and Mapllc3a / b (Data not shown). To further explore these findings, we sought to identify direct genomic targets of Foxkl in T cells. First, we performed chromatin immunoprecipitation coupled with sequencing (ChlP-seq) in the BI-141 T cell line expressing a tagged version of Foxkl (Foxkl-Flag) (Data not shown). ChlP- seq analysis with anti -Flag or anti-Foxkl antibodies led to the identification of 296 shared peaks mainly localized in gene promoters (Data not shown). The sequences of these peaks were enriched for a Foxkl consensus binding motif previously identified in non-immune cells (Data not shown). Interestingly we found, that this set of genes with Foxkl bound promoters was enriched for genes in the KEGG autophagy pathway (Data not shown). Consistently, genes associated with these enriched terms were also up-regulated transcripts in 6h-activated Foxkl- deficient T cells (Data not shown). Furthermore, ChlP-seq analysis revealed additional autophagy genes (Beas 3, Bnip3l, Ccpgl and Calcocol) not listed in the KEGG pathway, as well as the E3 ubiquitin ligase F-Box Protein 32 (Fbxo32') involved in muscle cell atrophy, all upregulated in Foxkl- / - T cells16(Data not shown). We next sought to validate our findings in primary CD4+ T cells for a subset of these gene promoters using qPCR-based ChIP quantification. In these conditions, we confirmed the DNA binding of Foxkl on promoters of Calcocol, Wipi2, Wdr45, Taxlbpl and Fbxo32, and its increase following T cell activation (Fig. 4). Importantly, comparative analysis of the protein abundance of these target genes when detected in the proteome between 24h-activated Foxkl -deficient and control CD4+T cells, indicated that the transcriptional repression mediated by Foxkl is also echoed at the protein level (Data not shown). Collectively, these data demonstrate that Foxkl represses autophagic programs during the first hours of T cell activation, thereby enabling the triggering and sustaining of expression of proteins involved in energetic processes. Discussion

[0052] The present study reports the critical role of Foxkl in T cell effector functions and differentiation. We found that, shortly after TCR stimulation, Foxkl translocates into the nucleus in an AKT-mTOR axis-dependent manner and binds to the promoters of autophagy genes, leading to their repression. This finding reveals Foxkl as an important molecular player acting downstream of mTOR and involved in the transient repression of the autophagy flux operating during the initiation of T cell responses23. Such a mechanism is essential to trigger the subsequent metabolic reprogramming of T cells to a level allowing full differentiation into effector cells24Consistent with T cell phenotypes reported following inhibition of mTOR signals by rapamycin 3 or loss of RAS homolog enriched in brain (RHEB)25, Foxkl -deficient T cells showed a reduced propensity to differentiate into effector cells and reduced effector functions but remained potent to acquire a long-lived memory-like phenotype. The observed reduction in effector functions resulted in a lower capacity to kill tumor cells and control tumor growth. Conversely, enforced expression of Foxkl in CD4+ and CD8+ T cells is sufficient to enhance effector cell differentiation and functions.

[0053] In naive T cells, metabolic processes are under the control of a high autophagy flux23‘24This flux is imposed by the low expression level of nutrient transporters that characterize naive T cells, and enable constant recycling of the intracellular metabolites necessary for survival. Indeed, autophagy induced by amino acid starvation in T cells selectively targets nutrient transporters for degradation, leading to reduced glycolysis. The autophagy genes repressed by Foxkl could be the core mediator of autophagy flux in naive T cells. Thus, to trigger the rapid differentiation into effector cells, one of the first events induced by TCR stimulation must lead to the termination of this flux, even before translation of nutrient transporters and glycolytic enzymes is initiated. A short lag of time between the termination of the autophagy flux and the induction of protein expression is probably sufficient to prevent degradation of the reservoir of proteins involved in metabolic pathways, enabling their accumulation and promoting a positive feedback process2. Given the final impact of the mTOR axis, and Foxkl in particular, on T cell fate, it would be valuable to investigate how much the variation in the expression of metabolic players in these initial steps of activation, accounts for the efficiency of differentiation into effector or memory cells.

[0054] The characterization of Foxkl clearly draws a parallel with the Foxos TF, also involved in T cell differentiation. Although Foxos and Foxkl are both regulated by AKT, the operating mechanism differs significantly. Foxos are constantly active and localized in the nucleus of quiescent T cells. Upon TCR stimulation, activation of AKT induces their phosphorylation on Ser and Thr residues which promote their nuclear exclusion. Phosphorylation of Foxos provides docking sites for the 14-3-3 proteins, which, along with the masking of the Nuclear Localization Signal (NLS), results in their sequestration in the cytoplasm compartment, thereby suspending their transcriptional activity26, 27. In contrast, Foxkl has a subcellular localization symmetrically inverted to that of Foxos. Our data suggests that sequestration of Foxkl in the cytoplasm in resting T cells is operated by GSK3. Indeed, GSK3 is constitutively active in resting T cells and its inhibition by AKT following TCR stimulation could release the constraint exerted by GSK3 on Foxkl28, 29. Our kinase inhibition assays also indicated that additional events of phosphorylation mediated by mTOR on Foxkl could be necessary to enhance Foxkl nuclear shuttling. The mutational approach targeting Ser / Thr motif in Foxkl, showed that some phosphorylation sites might be more effective in regulating Foxkl subcellular localization. The impact of combined mutations suggests that GSK3 might have a prominent role in maintaining Foxkl in the cytoplasm of resting T cells. Although early studies in other cell types have observed phosphorylation-dependent localization of Foxkl, there is no clear consensus on the exact phosphorylation sites and associated kinases responsible for each modification10, 19, 20. The challenge arises from the numerous potential phosphorylation sites, their close proximity, and their ability to substitute for one another when mutated. Nevertheless, it appears that enforcing Foxkl nuclear localization is sufficient to enhance T cell effector functions presumably through its transcriptional repression activity. Further investigation will be required to clarify how phosphorylation regulations modify Foxkl localization and functional activities. Finally, noting that the Foxkl binding motif and its targets differ from those of Foxos, it is tempting to speculate that the mTOR-AKT axis mediates a synergistic cooperation of these transcription factors in order to induce full T cell effector differentiation.

[0055] Overall, our results provide a basis for further investigation into how Foxkl control T cell differentiation and function in diverse physiological or pathological contexts. Since the AKT- mTOR axis integrates signals from contextual receptors (co-stimulatory and co-inhibitory receptors), cytokine receptors and nutrient sensors, it will be important to identify Foxkl- dependent and -independent pathways in these different contexts. Furthermore, these findings identify Foxkl as a potential novel target for modulating T cell effector functions in immunotherapeutic applications, such as those using engineered tumor-infiltrating lymphocytes and CAR-T cells. REFERENCES:

[0056] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method of treating an inflammatory autoimmune diseases in patient in need thereof comprising administering to the patient a therapeutically effective amount of a Foxkl inhibitor.

2. The method according to claim 1 the Foxkl inhibitor of the present invention is suitable for reducing proliferation and cytokine secretion of T cells, in particular autoreactive T cells, following TCR stimulation.

3. The method according to claim 1 or 2 wherein the Foxkl inhibitor is an inhibitor of Foxkl expression.

4. The method according to claim 3 wherein the inhibitor of expression is an antisense oligonucleotide that blocks the translation of Foxkl mRNA.

5. A method for screening a plurality of test substances useful for the treatment of an inflammatory autoimmune disease in a patient in need thereof comprising the steps consisting of (a) testing each of the test substances for its ability to inhibit the activity or expression of Foxkl and (b) and positively selecting the test substances capable of said inhibition.

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