WO2019175332A1 - Bh4 pathway inhibition and use thereof for treating t-cell mediated autoimmune diseases or hypersensitivity - Google Patents

Abstract

The present invention relates to a method of inhibiting T cell activity comprising inhibiting BH4 biological activity in said T cell, ex vivo and in vivo, in particular in the treatment of a T cell-mediated autoimmune disease or T cell-mediated hyper-sensitivity, as the invention acts against sensitized T cells. The invention further relates to kits for such methods.

Description

BH4 PATHWAY INHIBITION AND USE THEREOF FOR TREATING T-CELL MEDIATED

AUTOIMMUNE DISEASES OR HYPERSENSITIVITY

The present invention relates to the field of BH4 pathway modulation in cells.

Background of the invention

Tetrahydrobiopterin (in field abbreviated as "BH4"; and sold under the international nonproprietary name sapropterin, trade name KUVAN®) is an essential co-factor for several critical en zymes, including nitric oxide synthases, aromatic amino acid hy droxylases (phenylalanine, tyrosine and tryptophan hydroxylases) and the alkylglycerol mono-oxygenase. Through these enzymes, BH4 is required to produce nitric oxide (NO) , catabolize phenylala nine, synthesize the neurotransmitters dopamine, norepinephrine, epinephrine and serotonin, and to metabolize ether lipids. These functions made the BH4 pathway a pharmacological target in the past .

The BH4 pathway is summarized in Werner et al . , Biochem. J. 438, 397-414 (2011). BH4 biosynthesis involves actions of the enzymes GTPCH 1 (GTP cyclohydrolase 1, also "GCH1"; which typi cally is the rate-limiting enzyme for BH4 biosynthesis) , PTPS (protein tyrosine phosphatase, also termed "6- pyruvoyltetrahydro-pterin synthase" or "PTP") , and SR (se- piapterin reductase, also "SPR") . An alternative to SR involves action of CR (Aldo-keto reductase family member C3) and AR (Al- do-keto reductase family member BIO) . In addition, a BH4 salvage pathway involves action of DHFR (dihydrofolate reductase) . PCD (Pterin-4-alpha-carbinolamine dehydratase, also PCBD1) and DPR (dihydropterine reductase) are enzymes that synthesize BH4 from its metabolites in a recycling action.

Modulation of the BH4 pathway is known in the art. Various inhibitors or antagonists to these enzymes exist for negative modulation, also referred to as BH4 biological activity inhibi tion.

For example, BH4 biological activity inhibitors are dis closed in WO 2005/048926 A2, mainly for the purpose of reducing pain. In particular, compounds and composition that reduces the tetrahydrobiopterin (BH4) biological activity are disclosed, in cluding inhibiting sepiapterin reductase (SPR) , Pyruvoyltetrahy- dropterin Synthase (PTPS) , GTP cyclohydrolase (GTPCH) , Pterin- 4a-carbinolamine dehydratase, and dihydropteridine reductase (DHPR) by disclosed compounds.

GTP cyclohydrolase inhibitors and sepiapterin reductase in hibitors are disclosed in US 5,877,176, mainly to treat chronic inflammation from allograft rejection related to nitric oxide production .

Sepiapterin reductase (SPR) inhibitors in particular are disclosed in WO 2011/047156, WO 2016/069847 A1 and US

2017/307591 Al, such as the widely used compound SPRi3. WO

2016/069847 Al further discloses screening methods to identify further SPR inhibitors.

WO 2017/059191 Al discloses further SPR inhibitors, includ ing sulfasalazine and other sulfa drugs mainly for use in treat ing pain, inflammation, immunological disorders allegedly in cluding Parkinson's disease.

Chidley et al . , Nature Chemical Biology 7.6 (2011) : 375, de scribes the development of a yeast three-hybrid system for iden tifying drug-protein interactions. SPR is identified as a target of the anti-inflammatory drug sulfasalazine and its metabolites, sulfapyridine and mesalamine.

US 2016/031812 Al discloses small molecule heterocyclic SPR inhibitors and their uses for the treatment or prevention of various forms of pain, namely inflammatory pain, nociceptive pain, functional pain, and neuropathic pain.

Further GCH1 inhibitors and methods for GCH1 inhibitor screening are disclosed in WO 2011/035009 Al .

Previously unrelated to the BH4 pathway, T cell-mediated au toimmune disease is a class of autoimmune disease with a break down in immune tolerance in which T cell dysregulation or mal function plays a critical role in the pathogenesis of these dis orders. T cell-mediated autoimmune disease are for example type 1 diabetes mellitus, multiple sclerosis and other disorders with Type IV hypersensitivities. Common treatments aim at the antigen underlying the failing immune tolerance or symptom treatment like supplementing factor that is down-regulated due to autoim munity reactions, such as insulin in case of diabetes.

Latremoliere et al . , Neuron 86.6 (2015): 1393-1406, relates to BH4 pathway inhibition for treating neuropathic and inflamma tory pain.

Ziegler et al . Biochemical and Clinical Aspects of Pteri- dines 2 (1983) : 185-193 describes the screening of intermediates of the pterin metabolism for their action during concanavalin A- induced lymphocyte activation.

WO 96/40246 A1 suggests a treatment using an antagonist of a receptor on a surface of a T cell which mediates contact depend ent helper effector functions, for example, an anti-gp39 anti body. WO 2015/054612 A1 suggests the treatment of T cell- mediated autoimmune disease with an interleukin-2-inducible T cell kinase (ITK) inhibitor. US 2006/134113 A1 suggest treating a T cell-mediated hypersensitivity with an anti-IL-6 receptor antibody .

There remains a goal to find more effective treatments of T cell-mediated autoimmune diseases.

Summary of the invention

The present invention is based on the discovery of a new regulatory mechanism in T cells involving the BH4 pathways. This discovery led to the development of new treatments of T cells in order to modulate T cell activity.

In a first aspect the invention provides a method of inhib iting T cell activity comprising inhibiting BH4 biological ac tivity in said T cell. Said T cells can be ex vivo (in vitro) or in vivo, especially in the treatment of conditions, diseases or disorders that are mediated by sensitized T cells.

Accordingly, the invention further relates to a method of treating a T cell-mediated autoimmune disease or T cell-mediated hypersensitivity comprising inhibiting (reducing) BH4 biological activity in said T cell (s) . Related thereto, the invention pro vides a BH4 biological activity antagonist for use in the treat ment of a T cell-mediated autoimmune disease or T cell-mediated hypersensitivity. Also related thereto, the invention provides a method of manufacturing a pharmaceutical composition that is ca pable of reducing BH4 biological activity or that comprises a BH4 biological activity antagonist for use in a treatment of a T cell-mediated autoimmune disease or T cell-mediated hypersensi tivity. In particular, the invention provides SPRi3 for use in the treatment in the treatment of colitis, asthma, psoriasis or multiple sclerosis; and related thereto is provided a method of manufacturing a pharmaceutical composition comprising SPRi3 for use in treating colitis, asthma, psoriasis or multiple sclero- SIS .

The invention further relates to a kit-of-parts comprising (i) a BH4 biological antagonist and (ii) a T cell culturing me dium and/or a T cell adsorbent and/or a T cell-specific drug de livery agent.

All aspects of the invention are related, the methods and products for use can utilize the inventive kit and the kit can be suitable for any of the inventive methods of uses. All de tailed descriptions of e.g. BH4 biological activity or antago nists, relate to preferred embodiments of all aspects of the in vention .

Detailed description of the invention

The present invention relates to a method of inhibiting T cell activity comprising inhibiting BH4 biological activity in said T cell. Such a method can be performed ex vivo and in vivo, in particular in the treatment of a T cell-mediated autoimmune disease or T cell-mediated hyper-sensitivity, as the invention acts against sensitized T cells.

The inventive treatment of T cells can be specific to T cells or systemic (in vivo) . Specificity however is preferred. While the inventors have found that new regulatory pathways in T cells benefit from inhibiting or reducing BH4 biological activi ty in said T cell in the treatment of T cell-mediated hyper sensitivity, as the invention acts against sensitized T cells, other effects in vivo may actually benefit from BH4 biological activity. For example, the T cell-mediated hyper-sensitivity or T cell-mediated autoimmune disease may be associated with an in flammation, including a T cell-mediated inflammation.

BH4 and BH4 biological activity agonists have antioxidant and anti-inflammatory effect (Foxton et al . , Neurochem Res

(2007) 32:751-756) . In a treatment of the present invention it may be desirable to maintain antioxidant effects and have the natural antioxidant activity of BH4 in vivo preserved. Such preservation of BH4 biological activity for antioxidant activi ty, in particular in the circulatory system, can be facilitated by supplying an BH4 biological activity agonist in addition to the BH4 biological activity antagonist (the latter to mediate the inventive effect on T cells) .

The present invention also relates to a method for reducing the immunoreactivity of T cells, comprising the reduction or in hibition of the BH4 biological activity of the cells. This can result in lowered immunoreactivity of the cells to an antigen. Such an antigen can be associated with the T cell-mediated hy per-sensitivity or T cell-mediated autoimmune disease, such as said disease being characterized by a T cell reaction or sensi tivity against a particular antigen as known in the art.

The T cells can be from a patient suffering from a T cell- mediated autoimmune disease or T cell-mediated hypersensitivity. The present invention relates to both, therapeutic treatments of the patient and non-therapeutic treatments of T cells ex vivo, e.g. in cell culture, e.g. to study T cells. On the other hands, a therapy may relate to a treatment of T cells in the patient, e.g. by treating the patient as such with a pharmaceutical com position, or by treating the T cells ex vivo (e.g. after isola tion from the patient or a healthy individual) and reintroduc tion of the T cells into the patient. According to the therapeu tic aspect, the invention provides a BH4 biological activity an tagonist for use in the treatment of a T cell-mediated autoim mune disease or T cell-mediated hypersensitivity. Such autoim mune diseases are also referred to as autoimmune disorders.

The language "autoimmune disorder" or "autoimmune disease" is intended to include disorders in which the immune system, in particular the T cells - especially activated T cells like CD4⁺ or CD8⁺ cells -, of a subject reacts to autoantigens or harmless antigens, such that significant tissue or cell destruction oc curs in the subject. The term "autoantigen" is intended to in clude any antigen of a subject that is recognized by the immune system of the subject, the term included antigens of. The terms "autoantigen" and "self-antigen" are used interchangeably here in. The term "self" as used herein is intended to mean any com ponent of a subject and includes molecules, cells, and organs. Autoantigens may be peptides, nucleic acids, or other biological substances .

The language "T cell-mediated autoimmune disorder" or "dis ease" is intended to include autoimmune disorders in which the reaction to self primarily involves cell-mediated immune mecha nisms, as opposed to humoral immune mechanisms. Thus, the meth ods of the invention pertain to treatments of autoimmune disor ders in which tissue destruction is primarily mediated through activated T cells. However, even though the methods of the in vention are intended for treatment of autoimmune disorders in which reaction to self is primarily mediated by cells other than B cells, the autoimmune disorders may be characterized by the presence of autoantibodies. Non-limiting examples of T cell me diated autoimmune disorders that can be treated by the methods of the invention include multiple sclerosis, diabetes type I, oophoritis, and thyroiditis.

The present invention further extends to diseases with T cell-mediated immune reactions, including inflammation, that target non-self (in the meaning of being expressed by the sub ject's organism) but harmless foreign antigens or cells in the subject. In a normal healthy person, such harmless foreign anti gens or cells would be subject to immune tolerance. Such harm less antigens or cells are for example of the gut microflora. T cell-mediated immune reactions against these occur for example in inflammatory bowel disease, especially Crohn's disease and colitis. Antigens of such harmless organisms are not self antigens (they are not expressed by the subject's organism) but microflora antigens. Further harmless antigens include drugs. Pathological conditions may include T cell-mediated hypersensi tivity to drugs.

T cell-mediated autoimmune diseases or hypersensitivities may include T cell-dependent inflammation T cell-mediated auto immune diseases. Preferred T cell-dependent autoimmune diseases or T cell-dependent hypersensitivities are selected from multi ple sclerosis, allergic contact dermatitis, (autoimmune) type 1 diabetes mellitus, rheumatoid arthritis, giant-cell arteritis, reactive arthritis, coeliac disease, Rasmussen's encephalitis, acute disseminated encephalomyelitis, Sjogren's syndrome, aller gic granulomatosis, including Churg-Strauss syndrome, Hashimo- to's thyroiditis (hypothyroidism), Graves' disease, idiopathic thrombocytopenic purpura, Addison's Disease, sarcoidosis, Wegen er's granulomatosis, autoimmune encephalomyelitis, oophoritis, microscopic colitis, uveitis, such as non-infectious uveitis, primary biliary cirrhosis, autoimmune hepatitis, ankylosing spondylitis, contact dermatitis, atopic dermatitis, chronic thy roiditis, an allergy, a T cell-mediated allergy, a T cell- mediated food allergy, T cell-mediated allergic contact dermati tis, graft versus host disease, transfusion-associated graft versus host disease, Heiner syndrome, T cell-mediated hypersen sitivity reactions to drugs, T cell-mediated skin inflammation.

Some of these diseases may have variants that are non-T cell related. In such a case, the invention preferably relates to T cell-mediated variants of these diseases, that is, they include an origin in T cell sensitivity against an autoantigen or harm less antigen tot which the T cells are insensitive in a healthy person .

In particular preferred are treatments of T cells from pa tients with a type IV hypersensitivity, colitis, asthma, contact dermatitis, or multiple sclerosis. Also preferred is a T cell- mediated skin inflammation and an allergy or a T cell-mediated hypersensitivity reactions to drugs. An allergy can be selected from a T cell-mediated food allergy, food protein-induced aller gy, allergic contact dermatitis, etc.. Preferred allergies are allergic airway inflammatory disease and T cell-mediated skin dermatitis .

The T cell-dependent autoimmune disease may be a type IV hy persensitivity. According to the Coombs and Gell classification system, four types of hypersensitivity reactions can be defined: Type I also referred to as classic immediate allergy reaction is mediated by Immunoglobulin E (IgE) class antibodies. Type II hy persensitivity mode of function is cytotoxic, its mechanisms de pend on antibodies of classes IgM or IgG and the Complement sys tem. Type III immune complex diseases also are induced via IgG and the Complement system. In contrast to the mentioned immedi ate Type reactions hypersensitivities of Type IV are delayed and T cells, not antibodies, act as mediators.

Accordingly, the present invention relates to the treatment of a disease, wherein the origin of the disease is mediated or caused by a Type IV hypersensitivity reaction and/or wherein the origin of the disease is mediated or caused by a T cell sensi tivity. Such a sensitivity is usually not found in a normal healthy person or is at least not pathological in the healthy.

Example symptoms of Type IV hypersensitivity reactions in clude mild ones like a runny nose, but also more severe condi tions like poison ivy rash, (contact-) dermatitis, hypersensi tivity pneumonitis or allograft rejection. Pharmaceutical treat ment varies, including over-the-counter or prescription cortico steroid preparations, injectable or oral corticosteroids, and Burrow' s solution, a preparation made of aluminium acetate dis solved in water. Clinically administered, corticosteroids sup press the immune system, Burrow' s solution has astringent and antibacterial properties. Therapies can cause side effects, no tably in case of prolonged usage. Such state of the art treat ments can be combined with the inventive treatment.

The first delayed type hypersensitivity reaction described used only the tuberculin antigen (tuberculin reaction) , but the definition was later expanded to include cell mediated reactions to other bacterial and viral antigens, responses to pure protein with adjuvant or haptens, and host responses to allografts. This reaction has been shown to be dependent on the presence of memory T cells. Both the CD4+ and CD8+ fractions of cells have been shown to modulate a response.

The term "chronic transplant rejection" is reserved for cas es where the rejection is due to a chronic immune response against the transplanted tissue. This often leads to the need of a new organ transplant after approximately 10 years. Graft ver sus host disease is a result of cellular immunity and is an ex ample of a delayed type hypersensitivity response. Similar to the graft vs. host disease form of cell mediated immunity are some autoimmune diseases: Hashimoto ' s thyroiditis, Sjogren's disease, adrenalitis, polymyositis, and pernicious anemia. The pathological picture is one of mononuclear cell infiltration and tissue destruction. Finally, it is necessary to view delayed type hypersensitivity not as an individual phenomenon but rather as a group of related responses to antigen. These include the tuberculin reaction, Jones-Mote reaction, contact hypersensitiv ity and graft rejection. These reactions can be further divided into CD4+ and CD8+ compartments and then subdivided on the basis of the T cells' cytokine secretion patterns.

The Type IV hypersensitivity is preferably selected from al lergic contact dermatitis, autoimmune myocarditis, autoimmune diabetes mellitus type 1, granuloma, peripheral neuropathy, Hashimoto ' s thyroiditis, inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), multiple sclerosis, rheumatoid arthritis, a Tuberculin reaction.

Delayed hypersensitivity Type IV reaction can be an inflam matory response that develops 24 to 72 hours after exposure to an antigen the immune system recognizes as foreign. The reaction is mediated by T cells rather than by antibodies. Helper T (Th 1) cells produce cytokines like interferon gamma, interleukin (IL)-2, and tumor necrosis factor-beta and promote a cell- mediated immune response. These can be an indicator for the in ventive treatment.

The present invention provides the BH4 biological activity inhibition for the therapy of ailments mediated or caused by Type IV hypersensitivity reactions. According to the invention, BH4 biological activity inhibition is also used for the treat ment (or prevention, prophylactic treatment) of diseases mediat ed or caused by a Type IV hypersensitivity reaction or for the treatment (or prevention) of a disease, wherein the origin of the disease is related to a Type IV hypersensitivity reaction. Preferably, the Type IV hypersensitivity reaction comprises a medical condition for example diseases, disorders or ailments, to be treated in the context of a Type IV hypersensitivity reac tion. In particular, the Type IV hypersensitivity reaction is of a Type IV hypersensitivity disease, or symptoms of the Type IV hypersensitivity reaction or disease. The embodiments, however, do not extend to the treatment of granulocyte mediated diseases, in particular type I or III hypersensitivity reactions, inflam mation or of oedemas.

In these embodiments "prevention" should not be interpreted as an absolute success in the sense that a patient can never de velop an associated disease, reaction or condition but as the reduction of the chance of developing the disease, reaction or condition in a prophylactic treatment. Prevention by prophylac tic treatment is to be understood in the sense of a reduction of the risk of development of Type IV hypersensitivity reaction as sociated diseases not as a total risk avoidance. E.g. sensitized T cells can be treated even before clinical symptoms of the dis ease occur. Such T cells may be detected or isolated from the patient before a treatment.

Preferably, the Type IV hypersensitivity reaction is a chronic hypersensitivity reaction or chronic disease. Therefore, the BH4 biological activity inhibition either alone or in combi nation with other drugs is an attractive option for chronic pa tients. In a special embodiment the hypersensitivity disease is a delayed Type IV reaction mediated by cells not by antibodies. The Type IV hypersensitivity reaction is in particular mediated or caused by T cells including CD8+ cells and/or CD4+ cells, in particular Thl and/or Th2 cells.

In further embodiments the formulation is used for treatment of hypersensitivity reactions from any one of contact dermati tis, atopic dermatitis, hypersensitivity pneumonitis, chronic transplant reaction, graft versus host disease, cell mediated autoimmune diseases Hashimoto ' s thyroiditis, Sjogren's disease, adrenalitis, polymyositis, or pernicious anemia.

The step of inhibiting BH4 biological activity in said T cell can be performed in vitro and/or ex vivo, preferably in isolated and/or purified T cells. This has the advantage that T cell can be specifically treated with the BH4 biological activi ty antagonist, having the benefit that in vivo - apart from the T cells - the BH4 biological activity can remain unchanged - or can even be increased, such as by administering a BH4 biological activity agonist, like BH4 itself or one of its metabolic pre cursors, like a substrate of any one of the enzymes in the BH4 synthesis pathway mentioned above, like sepiapterin, or any BH4 analogue or other BH4 biological activity agonist as described in US 3,557,106, US 7,601,717, US 2010/0016328 Al, US

2006/0194808 Al, US 2003/0077335 Al, US 2010/00099997 Al, U.S. Pat. Nos. 5,698,408; 2,601,215; 3,505,329; 4,540,783; 4,550,109; 4,587,340; 4,595,752; 4,649,197; 4,665,182; 4,701,455;

4,713,454; 4,937,342; 5,037,981; 5,198,547; 5,350,851;

5,401,844; 5,698,408, Canadian application CA 2420374, and US 8,324,210 (all incorporated herein by reference).

The patient or subject to be treated and/or from whom the T cells are derived or obtained, may be a mammal, preferably a hu man. The patient preferably has or is predisposed to any one of the diseases and conditions mentioned herein. In preferred em bodiments, the patient is a patient who has received T cells (preferably within the last 24 months, more preferably within the last 12 months, even more preferably within the last 6 months, yet even more preferably within the last 3 months, espe cially within the last months or even with the last two weeks or even within the last week) . Typically, the patient is in need of the inventive treatment.

Preferably, the patient is not suffering from pain, neuro transmitter dysregulation, nitric oxide dysregulation, a non-T cell-mediated inflammation, allograft rejection related to ni- trie oxide production, inflammation caused by induced nitric ox ide production in immune cells. As known in the state of the art, BH4 has an effect on nitric oxide production, especially by vascular cells and may have anti-inflammatory effects (see back ground section) . Various uses due to these effects have been proposed for BH4 or its inhibitors. The present invention does not extend to such prior uses but is related to the new mecha nism with regard to T cells as described herein. Preferably, the patient does not suffer from such ailments as described in the prior art. In other cases, the patient may suffer from such ail ments but the T cells of the patient are specifically targeted for a treatment, such as by an ex vivo treatment of T cells or by T cell specific drugs or compositions.

The T cells may be maintained ex vivo in a T cell culturing medium, such as serum-containing, serum-free and in particular serum-replacement media. A preferred medium comprises FCS (fetal calf serum) . In general, any medium suitable to maintain or pro liferate T cells as known in the art can be used.

In preferred embodiments, said T cell is a peripheral T cells, such as a CD4⁺ or CD8⁺ T cell. CD4⁺ cells may be Thl cells, Th2 cells, Thl7 or TH b helper cells or a combination thereof.

As has been shown in the examples, such T cells, in particular activated T cells, can be reduced in activity. This can result in the reduction of T cell-mediated immune responses in a T-cell mediated autoimmune disease or hypersensitivity. T cell precur sors or immature T cells, such as DN3 thymocytes are typically not significantly affected by the inventive treatment, which re duces unwanted side-effects of the inventive treatment. The in ventive BH4 biological activity reduction is typically specific to peripheral T cells, with regard to T cells and their precur sors in general.

According to the invention, inhibiting BH4 biological activ ity in said T cell can be done in vitro and/or ex vivo. These cells can be isolated and/or purified T cells, such as isolated cells from a patient. Isolation may be in a sample from the pa tient that comprises the T cells, such as a sample of peripheral T cells. Such a sample can e.g. be a sample of body fluids of the circulatory system, including blood or lymphatic fluid. The sample can be of a sample comprising mixtures of peripheral cells or be specific for T cells, with a high T cell count, as can be reached by purification. An example commonly used are PBMCs (peripheral blood mononuclear cells) , which comprise lym phocytes (T cells, B cells, NK cells) and monocytes. The T cells of the invention (peripheral T cells, e.g. as defined above, preferably CD4+ and/or CD8+ T cells) may be purified, e.g. to 0.1% or at least 1%, preferably at least 10% or even to 30% or more, such as at 90% or more (all % in percentage of cells in the sample) . Purification can be facilitated according to sur face markers, that can be used for binding a cell to a ligand of such surface markers, like CD4 or CD8. The surface markers may be found on the T cells of interest, and are preferably are spe cific to the T cells of interest, i.e. only minor amounts are found on other cells such that purification to the desired quan tity (% as above) is achieved. An adsorbent, herein also re ferred to as T cell adsorbent, may be used to this effect. The adsorbent may be on a solid surface to facilitate ease of puri fication, such as by a washing step to remove other cells.

The method may further comprise reintroducing or introducing the treated T cell into a patient, e.g. the same patient from whom the T cells have been obtained. Such reintroduction may be for the inventive treatment of a T cell-mediated autoimmune dis ease or T cell-mediated hyper-sensitivity or its prevention.

This allows removing sensitised T cells from the patient and re introducing desensitized T cells that have been treated accord ing to the invention. The inventive treatment can also comprise an introduction of T cells to the patient without prior isola tion, e.g. by donor T cells or prior stored T cells. Of course, immune compatibility (MHC) should be tested when using donor T cells .

The invention also comprises diagnosing a patient with a T cell-mediated autoimmune disease or T cell-mediated hyper sensitivity or a predisposition thereto and then treating the patient with a BH4 biological activity reduction, e.g. with an antagonist, in vivo or ex vivo (i.e. based on (re) introduction of treat-ed T cells) - or in other words, detecting a T cell- mediated autoimmune disease or T cell-mediated hypersensitivity or a predisposition thereto in a patient and treating the pa tient with a BH4 biological activity reduction, e.g. using the antagonist, in vivo or ex vivo (i.e. based on (re) introduction of treat-ed T cells) . The diagnosis or detection is not neces- sarily performed together with the inventive treatment. The in vention also relates to treating patients or T cells that have been diagnosed or detected. Said diagnosis or detection can e.g. be detecting T cells, preferably peripheral T cells as described above, especially CD4+ and/or CD8+ T cells that are the cause of a T cell-mediated autoimmune disease or T cell-mediated hyper sensitivity or predispose the patient thereto. Such T cells may be sensitized against an auto-antigen or a harmless antigen as described above. Such T cell may in turn be in need to a desen sitizing therapy according to the invention by reducing BH4 bio logical activity as described herein. Accordingly, the invention also provides treating T cells according to the invention (in a therapy, in vivo or ex vivo) , which can be T cells that are sen sitive against (or reactive to) an auto-antigen or a microflora antigen, preferably wherein the method comprises detecting said sensitized (or reactive) T cells in a patient or after isolation from the patient.

In the entire context of the present invention, BH4 biologi cal activity can be inhibited or reduced by using a BH4 biologi cal activity antagonist. The invention encompasses treating the T cell with an BH4 biological activity antagonist. Such an an tagonist can be an inhibitor of any one of the enzymes in the synthesis of BH4. In particular, the BH4 biological activity an tagonist can be selected from an sepiapterin reductase inhibi tor, GTP cyclohydrolase 1 inhibitor, protein tyrosine phospha tase inhibitor, aldo-keto reductase family member C3 inhibitor, aldo-keto reductase family member BIO inhibitor, dihydrofolate reductase inhibitor, pterin-4-alpha-carbinolamine dehydratase inhibitor, dihydropterine reductase inhibitor, or combinations thereof .

Such inhibitors are readily available in the prior art, as e.g. mentioned in the background section. Further, WO

2005/048926 A2 (incorporated herein by reference) discloses method of identifying such inhibitors. Any such inhibitor can be used according to the invention.

For example, the BH4 biological activity antagonist can be selected from a GTP cyclohydrolase I inhibitor selected from a substituted pyrimidine, preferably hydroxyl, amino or halogen substituted pyrimidine, in particular preferred 2, 4-diamino-6- hydroxypyrimidine, 2, 5-diamino-6-hydroxypyrimidine, 4, 5-diamino- 6-hydroxypyrimidine, 4 , 5-diaminopyrimidine, and 4 , 6-diamino-2- hydroxypyrimidine ; an oxidized pterin, preferably neopterin, xanthopterin, isoxanthopterin and biopterin; a reduced pterin, preferably 7, 8-dihydro-D-neopterin, (6R, S) -5, 6, 7, 8-tetrahydro-D- neopterin, 7 , 8-dihydrofolic acid and 5, 6, 7, 8-tetrahydrofolic ac id. Such compounds are disclosed in US 5,877,176 (incorporated herein by reference) . Such compounds are disclosed in US

5,877,176 (incorporated herein by reference). Any compound dis closed in US 5,877,176 can be used according to the invention.

The BH4 biological activity antagonist can be selected from a GTP cyclohydrolase I inhibitor selected GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula ( I ) :

tautomer, prodrug, or pharmaceutically acceptable salt thereof, wherein:

K^J, R , R³, and R⁴ are each, independently, II_* optionally substituted Cj^ alkyl, or R¹ and R², R² and l³, or R² and R⁴ combine to form a double loud,

R⁵, R⁶, and R⁷ are each, independently, 1:1 or optionally substituted

alkyl, and

wherein one and only one of R¹ and R². R² and R or R² and R⁴ combine to form a double toad, and

when R⁵, R⁶, and R⁷ are H, R¹ andB? combine to form a double bond .and R³ is H, or when II^s, R*, and R⁷ are H, R² and R¹ combine to form a double bond and R¹ is H, R⁴ is not -C¾C₆H₅, -CUWp-CA-CN), -CH^-C^-CHs), -CH₂CHCH₂, - CH₂C(=0)-(p-C*H4-OMe), -CH2CC ⁾)NH-(o-C6¾-0Et), -CIl2C(=O)NH-(2- methoxy-S-chloro-CeHa), -CHaC{-0)NH-(2-methylcydohexy 1), or -CH₂C(=0)N H- (p-C₆H₄-SO₂Caz.epane)).

or selected from a GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula (II-A) or Formula (II-B) :

or a tautomer, prodrug, or pharmaceutically acceptable salt thereof, wherein R¹, R² and R³ are each, independently, H or op tionally substituted Ci_ ₆ alkyl;

or selected from a GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula (III) :

tautomer, prodrug, or pharmaceutically acceptable salt thereof, wherein

X¹ is O or NR¹;

X² is O or NR²;

R¹ and R² are each, Independently, selected from II, or optionally substituted

C_M alkyl;

R³ is H, halogen, or NR⁸!⁹ _» or E^J combines with R⁴ to form an oxo group; and R combines with R¹ m R² to form a ON bond or R⁴ combines with R^J to form an oxo group;

R⁵, R^® R⁷, R⁸, and R⁹ are each, independently, H or optionally substituted C_S alkyl; and

when R⁵, 1⁶, and R⁷ are H, X^!is NR¹, R¹ and R⁴ combine to form a ON double bond, and X is NH, R³ is not H or Nlfc, and

when R\ R^f’, and R " are H, X'is NH, R³ combines with R⁴ to form an oxo group, and X² is NR². R² is nut H,

or selected from a GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula (IV-A) or Formula

tautomer, prodrug, or pharmaceutically acceptable salt thereof, or according to

tautomer, prodrug, or pharmaceutical ly acceptable salt thereof, wherein R¹. R², R³, R⁵, R⁶, and R^T are each, independently, H or optionally substituted iY„ alky 1.

or selected from a GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula (V-A) or For mula (V-B) :

tautomer, prodrug, or pharmaceutically acceptable salt thereof,

tautomer, prodrug, or pharmaceutical I y acceptable sail thereof, wherein each of R¹, R⁶, and R⁷, is II or optionally substituted C_l-f, alkyl. or selected from a GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula (VI) :

tautomer, prodrug, or pharmaceutically acceptable salt thereof, wherein each of R¹, R², l⁶, aid R⁷ is, independently, H or optionally substituted C Y* alkyl. These GCH1 inhibitors are disclosed in WO 2011/035009 A1 (incor porated herein by reference) . Any compound disclosed in in WO 2011/035009 A1 can be used according to the invention. WO

2011/035009 A1 further teaches methods for GCH1 inhibitor screening and identification, which can be employed according to the invention for selecting a GCH1 inhibitor.

The BH4 biological activity antagonist can be selected from a protein tyrosine phosphatase inhibitor selected from etidro nate, -Bromo-4-hydroxyacetophenone, 4- (Bromoacetyl) anisole .

Such compounds and other inhibitors are available at

www . selleckchem. com.

The BH4 biological activity antagonist can be selected from a sepiapterin reductase inhibitor selected from N- acetylserotonin, N-acetyldopamine, N-acetyl-m-tyramine, N- chloroacetyldopamine, N-chloroacetylserotonin, N- methoxyacetyldopamine and N-methoxyacetylserotonin . Such inhibi tors are disclosed in US 5,877,176 (incorporated herein by ref erence) . Any compound disclosed in US 5,877,176 can be used ac cording to the invention.

The BH4 biological activity antagonist can be selected from a sepiapterin reductase inhibitor selected from a structure of Formula (VI I ) ,

wherein

each or X ^: and X is. independent l N , ( - ! 1. or ( -halogen:

A is a single bond. C -O), or S€¾;

R¹ is CJ h yUR \ halogen amino. CN SO _jR ¹X XIISOuv ^{1 "}, N ! K/( O iR s \ or C(^_0)N(R^{i A}V ; each R ^L is, independently, H or optionally substituted ( Vi, alk> 1; n is 0, L or 2;

R~ is CH₂OR , optionally substituted

alkyl, optionally substituted C_3-9 cycloalkyk oppomliy sub ikuled ary l. optionally substituted hcicroe elyk or optionally substituted lieteruaryl;

R ^{·^} is 1 1 or optionally substituted C _fi alkyl;

R^M and R’¹⁴ are both II, or iV^A and R combine to form -O;

R^4A and R^4B are both l l, or R ^,A and R^1B combine to form =( ):

each of ^" and R^:" is, ir.dcpcnJcm , I k oplVnulh substituted C alk\ k optionally substituted C w cycloalky k optionally substituted alkaryl, or optionally substit t aikheteroaryk and

wherein when A is C(-O), R¹ is Oi l, R² is CH₂OMe, R ^!A. R^® R^4A, and R^® arc each Ik a:ul R is 1 1. R^e is not I k

Such inhibitors are disclosed in WO 2011/047156 (incorpo rated herein by reference) . Any compound disclosed in WO

2011/047156 can be used according to the invention. The inhibi tors of WO 2011/047156 are particularly preferred according to the invention, especially SPRi3. WO 2011/047156 further disclos es methods of identifying a sepiapterin reductase inhibitor that can be used according to the invention.

Further methods of identifying a sepiapterin reductase in hibitor are disclosed in US 2017/307591 A1 and WO 2016/069847 A1 (both incorporated herein by reference) . In addition to methods of identifying inhibitors WO 2016/069847 A1 also disclosed se lecting dosages for such inhibitors, that can be used according to the invention. Any compound and dosage disclosed in WO

2016/069847 A1 can be used according to the invention, especial ly inhibitor SPRi3 (Fig IB of WO 2016/069847 Al) . In particular preferred is the use of SPRi3 (or any other sepiapterin reduc tase inhibitor) in the treatment in the treatment of colitis, asthma, psoriasis or multiple sclerosis. SPR inhibitors SPRi3 and QM385 (2- (5-methyl-4- (4- (2, 2, 2-trifluoroethyl) piperidine-1- carbonyl) -lH-pyrazol-l-yl)pyrrolo [2, 1—f ] [1, 2, 4] triazin-4 (3H) -one are further disclosed in Cronin et al . Nature

2018 ; 563 ( 7732 ) : 564-568 (incorporated herein by reference). QM385 may or may not be used according to the invention. US 2016/031812 A1 discloses small molecule heterocyclic SPR inhibitors, which can be used according to the invention.

The BH4 biological activity antagonist can be selected from a sepiapterin reductase inhibitor like sulfasalazine or a sulfa compound, such compounds are disclosed in W02017/059191 A1 (in corporated herein by reference) and include a compound of formu la VIII-A or VIII-B recited directly below, or a pharmaceutical ly acceptable salt thereof:

(VIII-A), (VIII-B)

wherein:

=-=-= an optional double bond:

Z is CR¹ or NR¹, or if the double bond is present, then Z is CR¹ or N;

Y is NR² or Cl², or if the double bond is present, then Y is N or CR²;

X is N or CR^Sa;

R¹ and R^*, taken together with the atoms to which lhe\ arc attached Gppp a I-.5- h-. or 7-nicmbered ring; or

R¹ and R² are independently selected from the group consisting of H, Cy-salkyl, Cj-jcycloalkyl, C_j-jhaloatkyl, and halo;

R and R^5® are independently selected front the group consisting of H and C_halky!;

L is hc!croaryl-Co^alkylenc-, aryl-Co_-salkyIenc-, -S-Ci_-salkylcnc-aryl, -S-Ci_ salky lcne-hcleroary I, -C i .sal ky ienc-S - aryl , or -C i salt lenc-S -heteroaryl;

R³ and R⁴. taken together with nitrogen atom to which they are attached form a 3-, 4-,

5-, 6-. or 7-membered monocyclic ring or 6-, 7-, s-. h~, 10-, 1 1-, 12-, 13-, or 14-membered spirci, fused, and/or bridged polyeyeiie te.g.. bicydio ring: or

R³ is selected from the group con isting oi H. (^', _¾alkyl, Cj-iocycloalkyl, Ci- sJialoalkyl, aryl, heteroaryl, and tV- a Iky lone O.

R⁴ is selected from the group consisting of CVwtlkyl. Cvnbvelnalkyl, Ci. haloalkyl. aryl, hcteroaryl, aud C_halk lene-G; and

each G is independently selected from the group consisting of CN, aryl, heteroaryl, cycloalkyl, and helerocycloalkyl .

The sulfa compound may be a sulfa analogon of sulfasalazine such as disclosed in W02017/059191 A1. Any compound disclosed in W02017/059191 A1 can be used according to the invention.

Kynurenine metabolites are further inhibitors of sepiapterin reductase and hence inhibit BH4 production (Haruki, H., et al .

J. Biol. Chem. 291, 652-657 (2016), incorporated herein by ref erence) . Any kynurenine metabolites, in particular xanthurenic acid, N-acetylserotonin, kynurenic acid, 8-hydroxyquinaldic ac id, picolinic acid, 3-hydroxyanthranilic acid, and kynurenine can be used according to the invention.

The BH4 biological activity antagonist can be selected from a dihydrofolate reductase inhibitor selected from methotrexate, aminopterin, 10-propargyl-5, 8-dideazafolate; 2 , 4-diamino, 5- ( 3 ' , 4 ' -dichlorophenyl ) , 6-methylpyrimidine ; trimetrexate ; py rimethamine; trimethoprim; pyritrexim 5,10- dideazatetrahydrofdate ; 10-ethyl, 10-deazaaminopterin; or py rimethamine. Such inhibitors are disclosed in US 5,877,176 (in corporated herein by reference) and on www.selleckchem.com. Any such compounds disclosed in US 5,877,176 can be used according to the invention.

Further BH4 biological activity antagonists can be selected from a phenothiazine compound such as fluphenazine . Fluphenazine is preferably provided as a hydrochloride. In other embodiments, fluphenazine or phenothiazine compounds are not used as BH4 bio logical activity antagonists due to side effects.

A further BH4 biological activity antagonist of the inven tion is EGFR Inhibitor III, also referred to as CAS 733009-42-2 or N- (4- ( (3, 4-Dichloro-6-fluorophenyl) amino) -quinazolin- 6-yl ) -2- chloroacetamide .

The BH4 biological activity antagonist can be selected from an inhibitory nucleic acid against an enzyme in a BH4 synthesis pathway selected from sepiapterin reductase, GTP cyclohydrolase 1, protein tyrosine phosphatase, aldo-keto reductase family mem ber C3, aldo-keto reductase family member BIO, dihydrofolate re ductase, pter-in-4-alpha-carbinolamine dehydratase, dihydropter- ine reductase. An enzyme inhibitory nucleic acid (directed against these enzymes) , can be a siRNA, antisense RNA, shRNA or sgRNA (combined with CRISPR-Cas) . Such an inhibitory nucleic ac id may be expressed by a nucleic acid encoding the inhibitory nucleic acid. Nucleic acids are preferably RNA or DNA.

The BH4 biological activity antagonist can be selected from an antibody against any enzyme in a BH4 synthesis pathway se lected from sepiapterin reductase, GTP cyclohydrolase 1, protein tyrosine phosphatase, aldo-keto reductase family member C3, al- do-keto reductase family member BIO, dihydrofolate reductase, pter-in-4-alpha-carbinolamine dehydratase, dihydropterine reduc tase. Antibody technology is well known in the art and such an tibodies may be obtained e.g. by immunization of test animals. The antibody may be monoclonal or polyclonal; it may be any type of antibody or antigen binding portion thereof including IgG, IgA, IgD, IgE, IgM, Fab, Fab', F(ab)2, Fv, single chain anti body, cameloid antibody or nanobody, an antigen binding domain, etc.. The antibody may bind the active site of the enzyme or otherwise inhibit its function in the BH4 synthesis pathway (in hibitory antibody) .

The BH4 biological activity antagonist can be used with or provided in a pharmaceutical preparation. Preferably, the phar maceutical preparation is in the form of a formulation for topi cal or mucosal application, preferably lotions, cremes, oint ments, powders, coverings, patches, band-aids, sprays, disper sion media and gargles. The BH4 antagonist preparation is espe cially suitable for topical application to treat skin or mucosal symptoms of the hypersensitivity mediated disease. But also sys temic treatment, e.g. parenteral or oral (also for specific mu cosal treatment), is possible. In embodiments, the BH4 biologi cal activity antagonist is the single active agent in the compo sition.

A further embodiment is characterized in that the prepara tion is intended for oral intake, preferably in the form of pas tilles, tablets, troches, lozenges, pills, gums, powders or drinking solutions. Systemic or topical distribution of a BH4 antagonist can be facilitated by formulations and carriers known in the state of the art.

The preparation may also comprise pharmaceutical carriers, excipients, preferably polymeric excipients, or additives. The term "carrier" refers to a diluent, e.g. water, saline, excipi ent, or vehicle, with which the composition can be administered. For a solid or fluid composition the carriers or additives in the pharmaceutical composition may comprise Si0₂, Ti0₂, a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (poly- vidone or povidone) , gum tragacanth, gelatine, starch, lactose or lactose monohydrate, alginic acid, maize (corn) starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; a glidant, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin. Pref erably, the preparation comprises buffers or pH adjusting agents, e.g. selected from citric acid, acetic acid, fumaric ac id, hydrochloric acid, malic acid, nitric acid, phosphoric acid, propionic acid, sulfuric acid, tartaric acid, or combinations thereof. A BH4 antagonist can be in the form of a pharmaceuti cally acceptable salt, for example sodium salt, may also be used. Other pharmaceutically acceptable salts include, among others, potassium, lithium and ammonium salts. Preferred excipi ents are polymers, especially cellulose and cellulose deriva tives .

In a further embodiment the preparation comprises pharmaceu tical carriers, excipients, vectors, additives, or adjuvants, preferably of polymeric origin. Such carriers or vectors may comprise liposomes, nanoparticles or micelles, which are espe cially preferred in case of large antagonists, such as inhibito ry nucleic acids or antibodies.

Preferably, a BH4 antagonist is formulated for administra tion in doses between 0.001 mg/kg body weight of a patient and 500 mg/kg, preferably between 0.1 mg/kg and 100 mg/kg, most pre ferred between 1 mg/kg and 40 mg/kg. The present invention also provides for the use of the pharmaceutical preparations. The preparation is not limited for to be administered at the same time when a T cell-mediated hypersensitivity, such as a type IV hypersensitivity reaction, occurs but can also be used before or after the reaction, e.g. for prophylactic treatment, i.e. a treatment before an expected exposure to an immune stimulant to reduce the force of the reaction.

The inventive BH4 biological antagonist can be delivered formulated and/or used with a T cell-specific drug delivery agent. The T cells are as described above preferably peripheral T cells.

Cell specific drug delivery can be facilitated by binding the therapeutic agent, the BH4 biological antagonist, together with an agent that binds the target T cells. Such a cell binding agent is e.g. an aptamer as described in Zhou et al . (Oligonu cleotides. 2011 Feb; 21(1): 1-10) for this purpose, or an anti- body, e.g. as described above, including any type of antibody or antigen binding portion thereof including IgG, IgA, IgD, IgE,

IgM, Fab, Fab', F(ab)2, Fv, single chain anti-body, cameloid an tibody or nanobody, an antigen binding domain, etc... Zhou et al . described not only aptamers, but also means to bind thera peutic agents (here: BH4 biological activity antagonists) to the cell-binding agents, such as be linkers, that may be covalent or non-covalent, such as by coordination binding or hybridization. Particularly preferred are T cell binding agents that bind a surface marker of a T cell, such as CD4 or CD8. Zhou et al de scribe CD4-specific aptamers that are particularly preferred ac cording to the invention. There reach high rates of internaliza tion of the therapeutic agent.

The present invention also provides a kit or kit-of-parts suitable in a use according to any one of claims 1 to 12 com prising (i) a BH4 biological antagonist and (ii) a T cell cul turing medium and/or a T cell adsorbent and/or a T cell-specific drug delivery agent. These components (i) , any of (ii) are de scribed above herein.

The present invention is further illustrated by the follow ing figure and example, without being limited thereto.

Figures :

Figure 1. GCH1/BH4 pathway is indispensable for effective T cell proliferation, a, Gchl-Gfp expression in 24 hour-activated

(CD62L¹⁰) CD4⁺ T cells after anti-CD3/CD28 stimulation, b, Dose- response of anti-CD3/CD28 stimulation of purified CD4⁺ Gchl-Gfp T cells, c, Immunoblot of GCH1 after 24 hour-TCR stimulation in CD4⁺ T cells, d, e, BH4 production upon 24 hour- anti-CD3/CD28 stimulation in purified CD4⁺ T cells (d) and in Gchl-null cells (e) . Data are shown as means ± s.e.m. *P < 0.05; **P < 0.01 (Student's t-test) f, Representative FACS blot (left panel) de picting early activation markers (CD25, CD62L) and IL-2 secre tion (right panel) before and after T cell stimulation (24 hours) . Data are shown as means ± s.e.m. NS, not significant (Student's t-test) . g, h, CD4⁺ and CD8⁺ T cell proliferation af ter 3 days from control and Gchl ;Lck mice. Representative exper iment from >15 experiments, i, CD4⁺ T cell proliferation after 3 days from control and Gchl;RORc mice. Representative experiment from >4 experiments. Top panels show representative FACS prolif eration traces, bottom panels bar show % of proliferating cells. Figure 2. Blockage of GCH1/BH4 abrogates T cell-mediated autoimmunity. a, b, Transfer colitis model of intestinal autoimmunity. a, Schematic outline (top) and colitis scores of transferred control and Gchl-ablated CD4⁺ T cells into Ragl^/ hosts. Data are shown as means ± s.e.m. n = 10 for each genotype ***P < 0.001 (Two-way ANOVA analysis, Dunnett's multiple comparisons test). b, Representative immunofluorescence depicting intestinal infil tration of various immune cells (CD4⁺ and CD3⁺ T cells, CDllc⁺ dendritic cells and MPO⁺ neutrophils) . Scale bar, 200pm. c, Al lergic airway inflammatory disease model and quantification of inflammatory cells in bronchoalveolar lavage fluids (BALFs) . Da ta are shown as means ± s.e.m. n=35 for control mice; n=31 for Gchl;Lck mice. *P < 0.05; **P < 0.01 (Student's t-test) . d, Per centage increase of ear swelling after re-challenge using the

2 , 4 , 6-trinitrochlorobenzene (TNCB) -dependent skin hypersensitiv ity model. Data are shown as means ± s.e.m. n=8 for control mice; n=9 for Gchl;Lck mice. ***P < 0.001 (Student's t-test). e, f, EAE model of CNS autoimmunity. Data are shown as means ± s.e.m. e, EAE scores of control and Gchl ;Lck mice, n = 6 for each genotype. ****P < 0.0001 (linear regression analysis was performed on the slope of each disease curve) . f, Mean maximal EAE severity in control and littermate Gchl;Lck mice. *P < 0.05 (Mann-Whitney test) .

Figure 3. Pharmacological inhibition of the BH4 pathway ameliorates T cell mediated inflammation, a, BH4 production in 24 hour-activated CD4⁺ T cells treated with DMSO vehicle or SPRi3 (50mM) . Data are shown as means ± s.e.m. *P < 0.05 (Student's t- test) . b, Representative 3 day-T cell proliferation histogram of wild type T cells stimulated with anti-CD3/anti-CD28 antibodies and treated with vehicle or SPRi3 (50mM) . Experiment was repeat ed >5 times showing similar results, c, d, Representative FACS blots depicting EdU cell-cycle analysis after 28hours anti- CD3/CD28 stimulation of control, Gchl ;RORc, and SPRi3-treated control CD4⁺ T cells. EdU was pulsed for the last 4 hours, d, Quantification of S-phase entry. Data from individual mice are shown ± s.e.m.. ***P < 0.001 (One-way ANOVA with Dunnett's mul tiple comparisons test) . e, Quantification of subGl (dead cells) populations after 24- and 48-hour stimulation. EdU was pulsed for the last 4 hours of each time point. Data from indi vidual mice are shown ± s.e.m.) . **P < 0.01; NS, not significant (Multiple t-test comparisons) . f, Colitis model of transferred control CD4⁺ T cells into Ragl^/ hosts treated with vehicle or SPRi3 (300mg/kg) . Vehicle or SPRi3 was administered for each week for 5 consecutive days. Data are shown as means ± s.e.m. n = 8 for each genotype. *P < 0.05 (Two-way ANOVA analysis, Sid- ak' s multiple comparisons test). Right panels show representa tive images of intestinal immune infiltration (CD4⁺ T cells, CDllc⁺ dendritic cells and MPO⁺ neutrophils) . g, Allergic airway inflammatory disease model in control mice treated with SPRi3 (300mg/kg) or vehicle. Vehicle or SPRi3 was administered for three consecutive days after OVA aerosol. Quantification of in flammatory cells in BALFs are shown as means ± s.e.m. Data from individual mice are indicated. *P < 0.05; **P < 0.01 (Student's t-test) . h, i, Vehicle- and SPRi3-treated (50mM) (h) naive human

(n=4 donors) CD4⁺ T cell proliferation assays stimulated via their TCR (anti-CD3 and anti-CD28) or (i) effector human CD4⁺ T cells re-challenged via their TCR (anti-CD3 and anti-CD28) . Data are shown as means ± s.e.m. **P < 0.01; ***P < 0.001 (Student's t-test) .

Figure 4. GCH1/BH4 affects iron homeostasis and mitochondrial respiration, a, Western immunoblot of iron regulators in acti vated peripheral CD4⁺ T cells from control and Gchl ;Lck mice, b, Dose-dependent reduction of ferri-cytochrome-C (FICC) to ferro- cytochrome-C (FOCC) by BH4. The doses of BH4 and the Fe²⁺ peak are indicated, c, Total iron content from unstimulated and anti- CD3/28 stimulated CD4⁺ T cells from control and Gchl;Lck mice. Data are shown as means ± s.e.m. n = 4 mice for each genotype.

*P < 0.05; NS, not significant (Student's t-test with multiple comparisons) . d, ATP measurements in control and Gchl ;Lck CD4⁺ T cells left unstimulated or assayed at the indicated timepoints after T cell activation (anti-CD3/anti-CD28 ) . e, ATP measure ments of wild type CD4⁺ T cells treated with DMSO vehicle or SPRi3 (50mM) . Data in d, and e, are shown as means ± s.e.m. n =

3 for each genotype. *P < 0.05; **P < 0.01 (Student's t-test with multiple comparisons) . f, Oxygen consumption rate (OCR) in unstimulated and 10 hour anti-CD3/CD28-stimulated CD4⁺ T cells from control and Gchl ;Lck mice. Data from individual mice are indicated ± s.e.m. **P < 0.01 (Student's t-test). g, h, Repre sentative oxygen consumption traces from permeabilized cells from 10 hour activated CD4⁺ T cells from (g) control and Gchl ;Lck mice and ( ) activated wild type mice treated with vehicle or SPRi3 (50mM) . i, Relative Complex I and II activities (mean val ues ± s.e.m.) in 10 hour activated control cells treated with vehicle or SPRi3 (50mM) . *P < 0.05 (Student's t-test) . N.S., not significant, j, Representative FACS histogram depicting DHE (di- hydroethidium, superoxide ROS indicator) levels in unstimulated and 10 hour anti-CD3/anti-CD28 activated CD4⁺ T cells from con trol, GCH1 ;RORc mice as well as control cells treated with SPRi3 (50mM) . Experiments were repeated 3 times showing comparable re sults. k, Proliferation of wild type control and Gchl ;Lck CD4⁺ T cells and rescue assay with the superoxide scavenger NAC (N- acetyl-L-cysteine ; 500mM) . Representative proliferation histo grams are shown on the left and quantification (mean ± s.e.m.) on the right. ****P < 0.0001 (One-way ANOVA with Tukey' s multi ple comparison test) .

Figure 5. Enhanced BH4 production results in T cell hyperproliferation . a, Representative immunoblot to detect GCH1 and the HA tag in naive CD4⁺ T cells from control and GOE;Lck overex- pressor mice, b, Fold change of BH4 levels and c, representative histograms after CD4⁺ T cell activation (ant-CD3/anti-CD28 ) of control and GOE;Lck mice. Experiments were repeated >3 times showing comparable results, d, Representative histograms depict ing dose-dependent proliferation of anti-CD3/CD28-stimulated CD4⁺ T cells for 3 days from control and GOE_/CD4 mice. Experiments were repeated >3 times showing comparable results, e, IL-2 and IFNy secretion after three days of CD4⁺ T cell activation (anti- CD3/anti-CD28 ) from control and GOE_/CD4 mice. Data are shown as means ± s.e.m. n = 3 for each genotype. *P < 0.05; ***P < 0.0001 (Student's t-test) . f, BH4 production after 24 hours in anti- CD3/anti-CD28 activated wild type CD4⁺ T cells treated with DMSO vehicle or sepiapterin (SP, 5mM) . Data are shown for individual mice as means ± s.e.m. ***P < 0.001 (Student's t-test). g, Rep resentative histograms depicting purified CD4⁺ and CD8⁺ wild type and Gchl ;RORc T cell proliferation after 3 days treated with SP (5mM) . The profile for the unstimulated T cells of each genotype is shown. Experiments were repeated 3 times showing comparable results, h, Effect of BH4 on IL-2 secretion (top) and T cell proliferation (³H thymidine incorporation; bottom) of CD4⁺ wild type T cells activated with anti-CD3/anti-CD28 and treated with vehicle or BH4 (10mM) . Data are shown for individual mice as means ± s.e.m. **P < 0.01 (Student's t-test) . i, Representative FACS histogram depicting DHE (superoxide ROS indicator) levels in unstimulated and anti-CD3/anti-CD28 activated CD4⁺ T cells for 10 hours from control and GOE CD4 littermates as well as wild type control cells treated with SP (5mM) . Experiments were re peated 3 times showing comparable results.

Figure 6. Modulation of BH4 enhances anti-cancer immunity, a,

Orthotopic breast cancer model in control (n=6) and GOE;Lck (n=7) mice. E0071 (2.5x10^s cells/200yL/mouse) tumor cells were injected into the mammary fat pad of syngeneic C57B16 mice and tumor sizes monitored over time, b, Effect of BH4 supplementa tion on breast cancer growth. E7001 mammary tumor cells were or- thotopically injected and once the tumors were palpable (day 10), BH4 (lOOmg/kg) or vehicle (saline) were therapeutically ad ministered. BH4 and vehicle supplementation was carried out for 7 days. Data are shown for individual mice as means ± s.e.m. **P < 0.01; * * * P < 0.001; ****P < 0.0001 (Student's t-test with mul tiple comparisons) . c, Quantification of intratumoral effector T cells (CD44⁺CD62L^l0) assayed from E0071 tumors on day 28 of vehi cle and BH4 treated mice. Data are shown as means ± s.e.m. NS; not significant, *P < 0.05; ****P < 0.0001 (2-way ANOVA with Sidak' s multiple comparison test), d, Representative histograms depicting 3-day proliferation of anti-CD3/anti-CD28 activated wild type CD4⁺ T cells treated with vehicle or kynurenine (50mM) . e, Quantification of proliferating of anti-CD3/anti-CD28 acti vated CD4⁺ T cells treated with kynurenine (50mM) and BH4 (10mM) . Unstimulated T cells are shown as controls. Data are shown as means ± s.e.m. **P < 0.01; NS, not significant (One-way ANOVA with Tukey' s multiple comparison test), f, Representative FACS histograms depicting DHE (superoxide ROS) levels in anti- CD3/anti-CD28 stimulated wild type CD4⁺ T cells treated with ve hicle (DMSO) , kynurenine (KYN) alone (50mM) or KYN (50mM) plus BH4 (10mM) for 10 hours. Experiments were repeated 3 times show ing comparable results.

Figure 7. Normal T cell development in the absence of Gchl . a,

Percentage of GFP⁺ cells from 24 hour-PMA/ionomycin (50ng/ml each) stimulated purified Gchl-Gfp CD4⁺ T cells. b, Cell numbers of various immune populations in the thymus (left panel) and spleen (right panel) from control and Gchl ;Lck 8-week-old mice. Data from individual mice are shown as means ± s.e.m. NS, not significant (Student's t-test) . c, Representative histogram de picting the proliferation of DN3a thymocytes from control and Gchl ;Lck mice cultured on OP9-D11 stromal cells for 5 days, d, e, Representative FACS blot depicting the differentiation of DN3a thymocytes from control and Gchl ;Lck mice cultured on OP9- Dll stromal cells for 5 days into CD4⁺ and CD8⁺ cells (d) and quantification of the differentiated cell types from n=3 animals (e) . Data from individual mice are shown as means ± s.e.m. NS, not significant (Student's t-test). f, Representative histogram depicting proliferation of control DN3a thmyocytes co-cultured with OP9-DL1 stromal cells treated with control or sepiapterin (SP, 5mM) for 4 days, g, h, Thymocyte cell death induced by var ious stimuli over 24 hours (g) and 48 hours (h) . Anti-CD3 (0.5,

5 pg/ml) , Fas ligand (0.2, 2 pg/ml) , dexamethasone (Dex, 0.1,

0.5 pg/ml) and g-irradiation (1 Gray(Gy)). Data are shown as means ± s.e.m. n=3 for each genotype. NS, not significant (Stu dent's t-test) . i, Death by neglect of purified CD4⁺ T cells cul tured without stimulation for up to 56 hours. Data are shown as means ± s.e.m. n=3 for each genotype. NS, not significant (Stu dent' s t-test) .

Figure 8. GCH1/BH4 is dispensable for B cell development as well as LPS-induced proliferation and class switching, a, FACS blots from spleens of control and Gchl ;MB1 mice depicting B cell de velopmental populations, b, c, Representative FACS histogram de picting LPS (lpg/ml) -stimulated B cell proliferation from con trol and Gchl ;MB1 mice after 3 days (b) as well as wild type control B cells treated with vehicle (DMSO) or SPRi3 (50mM) (c) .

Shaded grey peaks represents unstimulated cells. FACS blots are representative of two independent experiments. n=3 mice per group, d, Class switch recombination. FACS analysis of splenic CD43 B cells from control and Gchl ;MB1 mice stimulated with LPS (20pg/ml) for 5 days inducing class switch recombination to IgG3. FACS blots are representative of two independent experi ments .

Figure 9. Ova immunization defect in T cell specific Gchl- ablated mice. a,b, Ova-immunisation of control and Gchl ;Lck mice. T cell-dependent IgG responses and T cell-independent IgM responses are shown two weeks after OVA immunization (100pg ova in 200pg alum) (a, b, left panels) as well as two weeks after re-challenge (a, b, right panels) . n=5 for control mice; n=6 for Gchl;Lck mice. Data are shown as means ± s.e.m. *P < 0.05; **P < 0.01; * * * P < 0.001; NS, not significant (Student's t-test with multiple comparisons) .

Figure 10. SPRi3 and SP treatment does not affect early activation marker profiles nor are toxic at the doses use. a, Repre sentative FACS blots depicting activation marker profiles of pu rified wild type control CD4⁺ T cells left unstimulated or anti- CD3/28-stimualted for 24 hours and treated with vehicle (DMSO) , SPRi3 (50mM) or SP (5mM) . b, Cell survival as defined by DAPI AnnexinV cells of purified CD4⁺ T cells stimulated for 24- and 48-hours with anti-CD3/28 and treated with vehicle (DMSO), SPRi3 (50mM) and SP (5mM) . c, Representative histogram depicting 3 days of proliferation of purified control wild type CD4⁺ T cells co-cultured with purified (CDllc⁺) splenic dendritic cells serv ing as antigen presenting cells (APCs) plus soluble anti-CD3 an tibody (lpg/ml) and treated with vehicle (DMSO), SPRi3 (50mM) or SP (5mM) . FACS blots are representative of three independent ex periments, all showing similar results.

Figure 11. Amino acid and neurotransmitter profiles are unaltered in stimulated CD4⁺ T cells from control and Gchl;Lck mice. a, b, Amino acid profiles in the supernatants (a) and the cell pellets (b) from 24-hour anti-CD3/CD28 stimulated CD4⁺ T cells from control and Gchl ;Lck mice. n=3 for each genotype. Data are shown as means ± s.e.m. c, Biogenic amine profiles in the cell pellets (upper panel) and supernatants (lower panel) from 24- hour anti-CD3/CD28 stimulated CD4⁺ T cells from control and

Gchl ;Lck mice. n=3 for each genotype. Data are shown as means ± s.e.m.

Figure 12. Superoxide levels are enhanced upon BH4 deficiency and reduced through its overproduction. Quantification of the mean fluorescent intensity (MFI) from the FACS histograms of DHE staining from 10 hour anti-CD3/CD28 stimulated CD4⁺ T cells from control, Gchl;RORc, GOE;CD4 as well as wild type control T cells treated with SPRi3 (50mM) and SP (5mM) . n=3 for each geno type/condition. Data are shown as means ± s.e.m. **P < 0.01; ***P < 0.001 (One-way ANOVA with Dunnett's multiple comparisons test) .

Figure 13. iNOS uncoupling is not responsible for the enhanced superoxide levels observed in BH4-deficient activated T cells. a, Intracellular iNOS expression in purified CD4⁺ control T cells left untreated or anti-CD3/CD28 (4mg/ml+2mg/ml) stimulated for 12, 24 and 72 hours, b, representative histogram showing iNOS expression in control and Gchl-ablated CD4⁺ T cells anti-CD3/CD28 (4pg/ml+2pg/ml) stimulated for 72 hours. Quantification of iNOS⁺ cells over time. n=3 for each genotype. Data are shown as means ± s.e.m.; NS, not significant (Student's t-test) . c, Nitrite measurements in the supernatant of stimulated cells in (b) . Per itoneal, thioglycollate-elicited macrophages stimulated with LPS (lOOng/ml) for 24 hours was used as a positive control. Data are shown as means ± s.e.m.; NS, not significant (Student's t-test) . Figure 14. Increased effector T cells in naive mice overexpressing Gchl. a, Proportion of splenic T and B cells as well as the proportion of CD4⁺ and CD8⁺ T cells among the splenic T cell (TOKb⁺) population from control and GOE;Lck mice. Data of indi vidual mice aged 8 weeks are shown as means ± s.e.m. NS, not significant. (Student's t-test) . b, Proportion of CD4⁺ and CD8⁺ T cells among the splenic T cell (TOKb⁺) population from control and GOE;Lck mice. Data of individual mice aged 8 weeks are shown as means ± s.e.m. NS, not significant. (Student's t-test). c, Representative dot blot depicting naive (CD44¹⁰; CD62L^hl) , memory (CD44^hl; CD62L^hl) and effector (CD44^hl; CD62L¹⁰) T cell markers from the spleen of control and GOE;Lck mice (left panel) . Right panel depicts the quantification of the various subtypes within each population. Data of individual mice are shown as means ± s.e.m. * * P < 0.01; ***P < 0.001; NS, not significant (Student's t-test) . d, Cell numbers of B cells, T cells as well as CD4⁺ and CD8⁺ T cells in the spleens of control and GOE_/CD4 mice. Data from individual mice are shown as means ± s.e.m. NS, not signif icant. (Student's t-test) . e, Proportion of CD4⁺ and CD8⁺ naive (CD44^l ; CD62L^hi) , memory (CD44^hi; CD62L^hi) and effector (CD44^hi; CD62L¹⁰) T cells in the spleens of naive control and GOE;CD4 mice. Data of individual mice are shown as means ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant (Student's t-test) .

Figure 15. Overactivation of the GCH1/BH4 pathway leads to enhanced T cell activation and proliferation, a, Representative histogram depicting CD4⁺ T cell proliferation after 3 days of an- ti-CD3/CD28 stimulation from control and GOE ERT mice treated with 4-hydroxytamoxifen (4-OHT, 0.5mM) to induce Gchl overex pression in vitro. b,c, Quantification of 4-QHT-treated CD4⁺ T cell proliferation (b) and cytokine secretion (c) after 3 days of anti-CD3/CD28 stimulation from control and GOE ERT mice. Data from individual mice are shown as means ± s.e.m. **P < 0.01;

***P < 0.001 (Student's t-test) . d, Schematic representation of the de novo and salvage arms of the BH4 pathway. Dotted arrow indicates non-enzymatic reactions while solid arrows indicate enzymatic reactions. GTP, guanosine triphosphate; PTPS, 6- Pyruvoyl tetrahydropterin synthase; DHFR, dihydrofolate reduc tase. e, f, Representative FACS blot of EdU cell-cycle analysis after 28-hours anti-CD3/CD28 stimulation of control, GOE; CD4, control treated with SP (5mM) , and GCHl;RORc treated with SP (5mM) CD4⁺ T cells (e) and quantification of the S-phase entry population (f) . EdU was pulsed for the last 4 hours of stimula tion. * * * P < 0.001 (One-way ANOVA with Dunnett's multiple com parisons test) . g, Representative histograms depicting prolifer ation of wild type control CD4⁺ T cells after 3 days of anti- CD3/CD28 stimulation which were vehicle (DMSO) treated or sup plemented with BH4 (10mM) from control and Gchl ;RORc mice. FACS blots are representative of two independent experiments.

Figure 16. Flyphenazine in vivo treatment, reduced GCH1 protein levels in a dose-dependent manner, a, b, after sciatic nerve in jury (SNI), GCH1 transcription increases and protein levels in crease. Treatment with fluphenazine hydrochloride reduces GCH1 protein levels in the injured sciatic nerve. Naive is baseline while SNI was performed for 3 days and then the injured sciatic nerve was extracted and western blot performed. Actin was used as a loading control and data quantified using image J. c, d, treatment of T cells with luM fluphenazine ("Flu" or "Fluphen") did not affect early activation markers but reduced prolifera tion. T cells were activated with plate bound anti-CD3 (lug/ml) and anti-CD28 (2ug/ml) . 24hours later, the cells were collected and activation upregulation (CD25) and downregulation (CD62L) was analysed by flow. Fluphenazine had no effect on viability of activation marker (left graph) . Proliferation was reduced with fluphenazine treatment and interestingly was completely rescued with sepipaterin, thereby bypassing GCH1.

Figure 17. Dose-response of fluphenazine hydrochloride on T-cell proliferation. Prolfieration was measurement using a cell tracer violet dye from Invitrogen to label the naive t cells with. When the cells activate and proliferate the dye gets diluted out thus rescuing the signal on FACS in proliferating cells.

Figure 18. EGFR III inhibitor in in vivo treatment reduced GCH1 protein levels, a, 3 days after sciatic nerve injury (SNI), GCH1 transcription increases and protein levels increase. Treatment with EGFR III Inhibitor reduces GCH1 protein levels in the in jured sciatic nerve, b, treatment of T cells with EGFR III inhibitor reduced T cell proliferation. T cells were activated with plate bound anti-CD3 (lug/ml) and anti-CD28 (2ug/ml) and proliferation was analysed as above. EGFR Inhibitor III recued the proliferation and interestingly was completely rescued with sepiapterin, thereby bypassing GCH1.

Example 1 : Materials and Methods

Mice. Mice expressing eGFP under the Gchl promoter were used to label cells that upregulate Gchl after T cell activation

(Latremoliere, A. et al . Neuron 86, 1393-1406 (2015)). Mice with a Cre-dependent GCHl-HA overexpression cassette to induce BH4 overproduction and Gchl floxed mice prevented BH4 production have been previously reported (Chuaiphichai , S. et al . Hyperten sion 64, 530-540 (2014)). For both gain- and loss-of-function experiments, we bred GCHl-HA and Gchl floxed mice to the T cell- specific lines LCK-Cre, CD4-Cre, RORgammact-Cre or the ubiqui tous tamoxifen-inducible Rosa26-CreERT2 animals and also to the B cell-specific line, MBl-Cre .

Compounds. Sepiapterin (SP, 11.225), tetrahydrobiopterin (BH4, 11.212) were purchased from Schircks Labs, Switzerland. For in vitro use, both SP and BH4 were dissolved in DMSO to a stock concentration of lOmM. SPRi3 has been previously developed and was used as instructed (Latremoliere et al . , supra) . For T cell assays, SP was used at a concentration of 5mM, BH4 at a concen tration of 10mM and SPRi3 at a concentration of 50mM unless oth erwise stated in the figure legends. For in vivo use, BH4 was reconstituted in sterile saline under argon gas. Kynurenine (# K8625) and NAC (# A9165) were purchased from Sigma.

Determination of BH4 levels. BH4 (tetrahydrobiopterin), and oxi dized biopterins (BH2 and biopterin, ) were determined by high- performance liquid chromatography (HPLC) followed by electro chemical and fluorescent detection, respectively, following an established protocol (Crabtree, M. J. et al . J. Biol. Chem. 284, 1136-1144 (2009)). Cell pellets were freeze-thawed in ice-cold resuspension buffer (50 mM phosphate-buffered saline, 1 mM di- thioerythriol , 1 mM EDTA, pH 7.4) . After centrifugation at

13,200 rpm for 10 min at 4°C, supernatant was removed and ice- cold acid precipitation buffer (1 M phosphoric acid, 2 M tri chloroacetic acid, 1 mM dithioerythritol ) added. Following cen trifugation at 13,200 rpm for 10 min at 4°C, the supernatant was removed and injected onto the HPLC system. Quantification of BH4 and oxidized biopterins was obtained by comparison with external standards and normalized to protein concentration, determined by the BCA protein assay (Pierce) .

Lymphocyte proliferation. T cells were purified from spleens and lymph nodes of mice using microbeads (CD4⁺; CD8⁺, naive CD4⁺, Mil- tenyi Biotec) . 96 U-shaped plates were coated with anti-CD3 (4pg/ml, Biolegend) with/without anti-CD28 (2pg/ml, Biolegend) at the indicated concentrations unless otherwise stated in the figure legends in PBS for 3 hours at 37°C. T cells were then plated at 10⁵ cells/well in IMDM+PenStrep+Lgly+10% FCS . Beta- mercaptoethanol was omitted. PMA (50ng/ml) and ionomycin

(50ng/ml) were also used to stimulate purified T cells for 24 hours. Purified and activated T cells were cultured for 24 hours and expression of activation markers (CD62L, CD25, CD44, CD69) were analyzed using Flow Cytometry and the supernatant was col lected in which IL-2 and IFN-g concentrations was measured using ELISA kits (Biolegend) . Purified T cells were also stained with the Cell Trace Proliferation Kit ( Invitrogen) , cultured for 3 days and proliferation was assayed by flow cytometry on viable cells (DAPI-negative) . In addition, purified T cells were cul tured with purified splenic dendritic cells and soluble anti-CD3 antibody (lpg/ml) for three days. Similarly, B cells were puri fied using microbeads (CD19⁺; Miltenyi Biotec) from the spleen, loaded with cell tracer, stimulated with LPS (lpg/ml) and ana lyzed for proliferation as described above. For class switch re combination experiment, CD43 B cells were isolated from spleens by MACS (Miltenyi Biotec) and stimulated for 5 days with LPS (20 pg/ml) to induce switching to IgG3. Percentages of switched B lymphocytes were assessed by flow cytometry

EdU staining. The cell cycle status of T cells was assessed us ing the Click-iT® EdU Flow Cytometry Cell Proliferation Assay (Invitrogen) . Briefly, purified CD4⁺ T cells were activated with anti-CD3 (4yg/ml) and anti-CD28 (2yg/ml) as described above. EdU was pulsed into the wells for 4 hours after 16hrs of stimula tion. The cells were prepared and stained with EdU as per the manufacturer's instructions.

Mitochondrial respiration and metabolomics . Mitochondrial res piratory parameters were measured with high-resolution respirom etry (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria) . Routine respiration was measured by incubating cells in a buffer containing 110 mM sucrose, 60 mM K-lactobionate, 20 mM K-HEPES, 10 mM KH2P04, 3 mM MgC12, 0.5 mM EGTA and 1 g/L fatty acid-free bovine serum albumin at 37°C (pH 7.2) . To assess Complex I- and Complex II-linked respiration, cells were permeabilized with digitonin [8 mM] . Complex I-linked State 3 respiration was in duced by addition of 5 mM glutamate/5 mM malate and ImM adeno sine diphosphate (ADP) . Complex II-linked State 3 respiration was induced with lOmM succinate after addition of the Complex I inhibitor rotenone (Ing/mL). Respiration rates were obtained by calculating the negative time derivative of the measured oxygen concentration. Oxygen consumption rates were measured using Sea horse technology. To measure ATP, purified T cells were either left unstimulated or stimulated with plate-bound anti-CD3

(4yg/ml) and anti-CD28 (2yg/ml) for the times indicted in the figure. ATP was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) . To determine ROS levels, puri fied T cells were activated with anti-CD3 plate-bound anti-CD3 (4yg/ml) and anti-CD28 (2yg/ml) for 10 hours. Cells were washed once with HBSS and stained in 10mM DHE (Invitrogen) for 30 mins at 37°C. Cells were washed 2X with HBSS and assayed by flow cy tometry. Profiling of biogenic amines by hydrophilic interaction liquid chromatography (HILIC-QTOF) mass spectrometry was per formed on cell pellets and supernatants from unstimulated and TCR-stimulated purified T cells by the West Coast Metabolomics Center (UC Davis) . For N0₂ ^~ measurements we used the Griess rea gent system (TB229, Promega) . Purified T cells were stimulated with anti-CD3 and anti-CD28 antibodies as described above and the supernatant was collected at various timepoints for nitrite measurements. Peritoneal macrophages stimulated with LPS

(lOOng/ml) for 24 hours were used as a positive control.

Mammary cancer orthotopic model. E0771 cells were orthotopically injected into syngeneic control and GOE;Lck mice as previously described (Ewens, A., et al . Anticancer Res. 25, 3905-3915 (2005)) . In brief, cells were harvested for injection into mice by trypsin digestion for 5 minutes, washed in Hank's Balanced Salt Solution, counted, diluted in this salt solution and ortho- topically injected into the fat pad of the fourth mammary gland (2.5x10^s cells/200yL/mouse) . BH4 administration was delivered i.p. (lOOmg/kg) after tumors were palpable (day 10) and treat ment was continued for 7 days. Tumors were measured using digi tal calipers; the size of the tumor was expressed as length (mm) x width (mm) x height (mm) = tumor size (mm³) .

Flow cytometry. Antibody labeling of cells was carried out in FACS staining buffer (PBS supplemented with 2% FCS and 2 mM EDTA) on ice for 30 min after blocking Fc receptors.

Table 1: List of antibodies (or their targets) used in this study .

antibody number company

Actm Sigma

AnnexinV BD

anti -CD28

Biolegend

anti CD 145-201 Biolegend

B220 RA3-6B2

CD21 7Gb

CD23 B3B4

CD25 7D4

CD4 RM4-5

CD 4 IM7

CD4S 30- 111

CD62L MCL-14

CDS 53.6-7

CD93 AA4.1

ill Trace Violet C3 557 T”ernoSci

DAPI Sigma

DHL

LdU

f-erntin

F-rataxin abll^‘3691 Abeam

GAPDI I 1400 Cel!Signaltng

GCH 1 GS Santa Cruz

HA H6908 Sigma

Hoeschst 62249 Tt-er oSci

HO-1 AD1- O5A -110 Lnzo

iWOS 610330 BD

|RGI AB5-1 BD

IRGB R40-82 eBio

itoferrin abl02959 Abeam

TCRbeta H57-597 BD

Cells were recorded on an LSR II flow cytometer (BD Biosci ences) , and data were analyzed using FlowJo vlO.O.6 software (Tree Star) . Absolute splenocyte and thymus numbers were deter mined by counting total cells with a CASY1 counter and subse quent calculation of T cell and B cell numbers based on ratios from FACS experiments.

Protein blotting. Protein blotting was carried out using stand ard protocols. Blots were blocked for 1 hour with 5% BSA in TBST (lx TBS and 0.1% Tween-20) and were then incubated overnight at 4°C with primary antibodies (See Table 1), diluted in 5% BSA in TBST (1:1,000 dilution) . Blots were washed three times in TBST for 15 min and were then incubated with HRP-conj ugated secondary antibodies (1:2,500 dilution; GE Healthcare, NA9340V) for 45 min at room temperature, washed three times in TBST for 15 min and visualized using enhanced chemiluminescence (ECL Plus, Pierce, 1896327) .

OP9-DL1 co-cultures. OP9 bone marrow stromal cells expressing the Notch ligand DL-1 (OP9-DL1; kindly provided by Juan Carlos Zhhiga-Pflucker ; University of Toronto) were maintained as de scribed previously (Schmitt, T. M., et al . Immunity 17, 749-756 (2002)) . 10⁴ OP9-DL1 were plated per well in 48 well plates 4-12 hours before the start of thymocyte cultures. DN3a thymocytes were sorted as

cells using a BD FACS Aria sorter. CellTrace Violet labeling of the sorted cells was performed in 1 mM CellTrace Violet solution in PBS containing 0.1% BSA for 7 min at 37°C. Cells were washed with me dium containing 20% FCS . Thymocytes were then plated on the OP9- DL1 monolayers in the presence of 5 ng/ml Flt3L. Co-cultures were performed in MEM supplemented with 10 mM HEPES (pH 7.5), 1 mM sodium pyruvate, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 20% heat inactivated FBS .

Adoptive transfer model of colitis. 5x10⁵ MACS-purified naive CD4⁺CD62L⁺ T cells from control and GCH1 ;Lck mice were injected i.p. into 6- to 8 weeks old Ragl ^/ mice. After the cell transfer, Ragl ^/ recipients were weighed weekly and monitored by mini endoscopy. For monitoring of colitis activity, a high-resolution video endoscopic system (Karl Storz) was used. To determine co litis activity, mice were anesthetized by injecting a mixture of ketamine (Ketavest lOOmg/ml, Pfizer) and xylazine (Rompun 2%, Bayer Healthcare) i.p. and monitored by mini-endoscopy at the indicated time-points. Endoscopic scoring of five parameters ( translucency, granularity, fibrin, vascularity and stool) was performed. For histological analysis, colonic cross sections were stained with H&E. Immunofluorescence of cryo-sections was performed using the TSA Cy3 system ( PerkinElmer) and a fluores cence microscope (1X70; Olympus) using primary antibodies against F4/80, MPO, CD3, CD4 and CDllc. In brief, cryo-sections were fixed in ice-cold acetone for 10 minutes followed by sequen tial incubation with methanol, avidin/biotin (Vector Laborato ries) , and protein blocking reagent (DAKO) to eliminate unspe cific background staining. Slides were then incubated overnight with primary antibodies specific for the respective antigen.

Subsequently, the slides were incubated for 30 minutes at room temperature with biotinylated secondary antibodies (Dianova) . All samples were finally treated with streptavidin-horseradish peroxidase and stained with Tyramide (Cy3) according to the man ufacturer's instructions (Perkin Elmer). Before examination, nu clei were counterstained with Hoechst 3342 ( Invitrogen) .

OVA immunization and Airway hyperresponsiveness. For OVA immun ization study, immunization was performed using lOOyg OVA per mouse in 200yL Alum intraperitoneally (i.p.). Blood was collect ed from the tail vein 14 days after injection to check IgG and IgM titers. 3 weeks later a further i.p. injection was carried out and again blood collected two weeks later to measure the re challenge responses. For measurements of lung function, deeply anesthetized mice (pentobarbital (60 mg/kg) underwent a trache otomy with a 20G sterile catheter. A computer-based analysis of airway hyperresponsiveness was then performed using a Flexivent (SCIREQ) apparatus. Mice were ventilated at a tidal volume of 9 ml/kg with a frequency of 150 bpm; positive end-expiratory pres sure was set at 2 cm H₂0. Lung resistance and elastance of the respiratory system was determined in response to in-line aeroso lized methacholine challenges (0, 1, 3, 10, 30, 100 mg/ml) .

Methacholine was dissolved in sterile PBS. The mean elastance and resistance of 10 measurements by doses was calculated. For bronchoalveolar lavage (BAL) on day 21, mice were anesthetized following an intraperitoneal injection of urethane (200 mΐ i.p., 35%) and a 20G sterile catheter inserted longitudinally into the trachea. 2 ml of ice cold PBS containing protease inhibitors (Roche) was injected into the lung, harvested and stored on ice. BAL fluid underwent a 400g centrifugation (15 min; 4°C) , the su pernatant was discarded and cells resuspended in 200 mΐ . Bron choalveolar lavage fluid (BALF) cells were resuspended in FACS buffer (PBS, 2% FCS, EDTA) , and incubated with Fc block (0.5 mg/ml, 10 min; BD Biosciences) . Cells were then stained with monoclonal antibodies (FITC anti-mouse CD45, BD Biosciences, cat no: 553079, PE anti-mouse Syglec-F, BD Biosciences, cat no:

552126; APC anti-mouse GR-1, eBiosciences , cat no: 17-5931-81; PE-Cy7 anti-mouse CD3e, cat no: 25-0031-81; PerCP anti-mouse F4/80, BioLegend, cat no: 123125; PE anti-mouse, BD Bioscience, cat no: 552126; 45 min, 4°C on ice) before data acquisition on a FACS Canto II (BD Biosciences) . A leukocyte differential count was performed during flow cytometry analysis of cells expressing the common leukocyte antigen CD45 (BD Pharmigen; cat no: 553079). Specific cell populations were identified as follows: macrophages as F4 /80^Hl-Ly6g^Neg, eosinophils as F4 /80^Int-Ly6g^Lo- SiglecF^Hl, neutrophils as F4 /80^Lo-Ly6g^Hl-SiglecF^Neg, and T lympho cytes as F4 /80^Neg-Ly6g^Neg-CD3^Pos . Total BAL cell counts were per formed using a standard hemocytometer, with absolute cell num bers calculated as total BAL cell number multiplied by the per centage of cell subpopulation as determined by FACS.

Skin hypersensitivity. The skin contact hypersensitivity model was performed as previously described (Martin, S. F. et al . J. Exp. Med. 205, 2151-2162 (2008) .) . Briefly, for the induction of contact hypersensitivity, mice were sensitized on day 0 by ap plying 100 mΐ of 7% 2 , 4 , 6-trinitrochlorobenzene (TNCB- Sigma) /acetone or acetone alone as vehicle control on the shaved abdomen. On day 5 mice were challenged on the dorsum of both ears with 20 mΐ of 1% TNCB/acetone . Ear thickness was measured immediately before and 24 hours after the challenge.

Experimental allergic encephalitis (EAE) . EAE was induced in control and Gchl ;Lck mice by immunization with an emulsion of lOOmg MOG_35-55 in complete Freund's adjuvant (CFA) , supplemented with 5mg/ml Mycobacterium tuberculosis (Difco) . 100 pL MOG/CFA was injected subcutaneously above the inguinal lymph node on both sides of the mouse. 200 pL pertussis toxin/PBS (50ng/mL- List Biological Labs) was injected intraperitoneally per mouse on day 0 and day 1. Scoring for EAE was performed as previously described over the course of 45days (Boivin, N., et al . PLoS One 10, (2015) ) .

Microarray analysis. Purified CD4⁺ T cells from control and Gchl ;Lck mice were stimulated with plate bound anti-CD3 (4pg/ml) and anti-CD28 (2pg/ml) for 16 hours and total RNA was extracted by sequential Qiazol extraction and purification through the RNeasy micro kit with on column genomic DNA digestion (Qiagen) . RNA quality was determined by an Agilent 2100 Bioanalyzer using the RNA Pico Chip (Agilent) . RNA was amplified into cDNA using the Ambion wild-type expression kit for whole transcript expres sion arrays, with Poly-A controls from the Affymetrix Genechip Eukaryotic Poly-A RNA control kit. Images from Agilent arrays were processed by Agilent Feature Extraction Software 10.7.3.1. Raw intensity data were processed in R v3.4.0 using limma v3.34.3 applying normexp background calculation, lowess within- array and Aquantile between-array normalization methods. The normalized values were used to calculate log2 transformed

Cy5/Cy3 ratios. Differentially expression analysis was performed by fitting a linear model to the normalized data and computing empirical Bayes test statistics in limma accommodating a mean- variance trend. False discovery rate was controlled by Benja- mini-Hochberg adjustment.

Ferric/Ferrous reduction. The enzymatic activity of BH4 was as sayed as previously described (Arai, N., et al . Pediatrics 70, 426-30 (1982)). The enzymatic conversion of qBH2 to BH4 was fol lowed by the reduction of Ferricytochrome-c (FICC) to Ferrocyto- chrome-c (FOCC) by BH4. FICC reduction was determined by reading the increasing FOCC absorbance signal at 550nm. The experiment was run for 40 minutes at pH 7.4 and recorded at 10 seconds in tervals in 200m1 buffer containing 50mM FICC, ImM 6- methyltetrahydropterin (6MPH4), 20nM DHPR, 50mM NADH and select ed inhibitors. A control lacking DHPR was ran in parallel to as sess the rate of non-enzymatic reduction of qBH2 by NADH. The extinction coefficient used for FOCC and NADH are respectively 29500 (reduced, 550nm, H₂0) and 6220 (340nm, ¾0) [L -mol ¹ -cm ¹] . Generally, 50mM of FOCC and FICC in buffer were measured in iso lated wells to assess completion of the reaction.

Iron Measurements. Total iron content was measured as previously (Theurl, I. et al . Nat. Med. 22, 945-951 (2016).). Briefly, in tracellular iron measurements were carried out by using a Perki- nElmer Analyst 800 equipped with a transversely heated graphite atomizer (THGA) . A Zeeman-effect background correction was real ized by a 0.8 T magnetic field, oriented longitudinally with re spect to the optical path. A PerkinElmer Lumina single-element iron hollow cathode lamp was driven at a constant current of 30 mA after proper equilibration (i.e., ³20 min) . For the absorp tion measurements, the 248.3-nm line (spectral bandwidth 0.7 nm) was chosen. FACS-purified naive CD4⁺ T cells from control and Gchl ;Lck mice were left untreated or stimulated (anti-CD3 and anti-CD28) for 12 hours. The cells were then pelleted and frozen at -80°C. Next, the samples were suspended in 200 mΐ of a 0.1% (v/v) solution of nitric acid (Rotipuran Supra, 69%, Carl Roth GmbH, Karlsruhe, Germany) in high-purity water (Milli-Q, Merck- Millipore, Darmstadt, Germany) by extended periods (i.e., ³30 min) of vortexing and ultrasonication at 30-40 kHz. After an in itial estimation of the sample's iron quantity, a five-point linear calibration was established in the range between 0 (i.e.,

<0.004 mM) and 0.106 mM. The calibration standards were prepared by diluting a 0.1 M standard stock solution of (NH_{4) 2}Fe (S0_{4) 2} (Merck-Millipore, Darmstadt, Germany) with a 0.1% (v/v) aqueous solution of nitric acid (vide supra) . The absence of detectable iron (i.e., <0.004 mM) in the dilution agent, as well as in the sample cups, and the glassware was verified throughout the anal yses. A linear fit of the 15 data points ( k = 0.978, d = 0.006 mM) yielded a coefficient of determination of 0.992. Samples with iron concentrations exceeding the calibration range (i.e., ³0.106 mM) were diluted appropriately. The blank solution, the calibration standards, and the samples were supplied to the at omizer in randomized fashion as triplicates, using a PerkinElmer AS-800 autosampler with an injection volume of 20 mΐ . The sol vent was evaporated by a slow temperature gradient to 130 °C, ashing took place at a maximum temperature of 1,000 °C, and the atomization profile was read at 2,000 °C. The graphite tube, which was protected against oxidation by high-purity argon

(99.999%, Messer Austria GmbH, Gumpoldskirchen, Austria), was cleaned out after each analysis at 2,450 °C. The integrity of each analysis was verified by a visual inspection of the respec tive time-dependent atomization profile.

Human T cell proliferation assays. Proliferation of peripheral blood mononuclear cells (PBMCs) , obtained from healthy blood do nors, was assessed following cell exposure to Dynabeads Human T Activator CD3/CD28 (bead/cell ratio: 1/2) and IL2 (30 IU/ml) , in the absence or presence of vehicle (DMSO) or SPRi3 (50mM) . PBMCs were resuspended in RPMI 1640 medium supplemented with 2mM L- glutamine, lOOU/ml penicillin, 100 mg/ml streptomycin, 1% non- essential amino acids and 10% FBS, seeded at 2.5xl0^s/well and cultured for 5 days. For the last 18 hours of culture, cells were pulsed with 0.25 mCi/well ³H-thymidine . Incorporated thymi dine was measured by liquid scintillation spectroscopy. In addi tion, we also determined the proliferation of alloreactive human T cells. PBMCs from a healthy donor were stimulated with M21 tu mor cells. Alloreactive T cells-based on MHC mismatch were cul tured for 2 weeks. Afterwards, effector CD4⁺ T cells were sorted (regulatory T cells excluded) , CFSE-labelled, and stimulated with anti-CD3 and anti-CD28 for 5 days with either DMSO or SPRi3 (50mM) supplementation. Statistical analyses. All values are expressed as means +/- s.e.m. Details of the statistical tests used are stated in the figure legends. Briefly, Student's t-test was used to compare between two groups. One-way ANOVA followed by Dunnett ' s post-hoc test for multiple comparisons was used for analysis between mul tiple groups. 2-way ANOVA was used to compare two groups over time. In all tests P £ 0.05 was considered significant.

Example 2: GCH1 controls T cell proliferation.

When findung GCH1 induction by T cell activation, we isolat ed CD4⁺ and CD8⁺ T cells from our Gchl-Gfp reporter mouse line (Latremoliere, A. et al . , Neuron 86, 1393-1406 (2015)), to re port transcriptional induction. Naive cells had no detectable GFP/Gchl expression in either cell type, but PMA and ionomycin increased GCH1 expression in activated T-cells (Extended Fig. la) . T cell receptor (TCR) stimulation using plate-bound anti- CD3 and/or anti-CD28 crosslinking antibodies induced GFP expres sion specifically in activated T cells (Fig. la) in a dose- dependent manner (Fig. lb). Gchl upregulation in anti-CD3/CD28- stimulated T cells was confirmed by Western blotting (Fig. lc) . GCH1 is the rate-limiting enzyme in the de novo production of the co-factor BH4 and anti-CD3/CD28-stimulated T cells produce large amounts of BH4 (Fig. Id) . Thus, GCH1 and BH4 are strongly induced in activated T cells.

To next explore the function of the GCH1/BH4 pathway in T cells, we generated Gchl T cell-specific knockout mice by cross ing Lck-Cre driver mice with Gchl (fl/fl) mice (Chuaiphichai , S. et al . Hypertension 64, 530-540 (2014)). Stimulation of CD4⁺ T cells isolated from these Gchl ;Lck mice did not increase GCH1 protein levels nor BH4 production unlike control CD4⁺ T cells (Fig. lc, e) , indicating that GCHl-dependent de novo synthesis is a primary source of BH4 in stimulated T cells. Gchl ;Lck mice showed normal numbers of thymic and peripheral T cell popula tions compared to Cre-only controls, revealing that GCH1 does not play an obvious role in T cell development or in peripheral T cell homeostasis in the spleen and lymph nodes (Fig. 7b) . This is in line with the observation that naive T cells, as well as thymocytes, from the Gchl-Gfp reporter line show no GFP expres sion (Fig. la) . As GCH1 and BH4 are induced upon TCR stimula tion, we evaluated antigen receptor signaling in mature periph- eral T cell activation. Following TCR engagement, we observed no differences between Gchl-null T cells and control cells in ei ther surface activation marker expression or in interleukin (IL)-2 secretion after 16 hours of TCR (anti-CD3/CD28) stimula tion (Fig. If), suggesting that early events in T cell activa tion are not dependent on the GCH1/BH4 pathway. Similar results were obtained in CD8⁺ T cells. However, antigen receptor- stimulated Gchl-deficient CD4⁺ and CD8⁺ T cells displayed a mark edly reduced capacity to proliferate (Fig. lg, h) . In contrast to peripheral T cells, Gchl ablation did not affect prolifera tion of DN3a thymocytes in response to signals from co-cultured OP9-DL1 stromal cells (Fig. 7c-f) , in line with the observed normal thymocyte development. Moreover, there were no obvious differences in survival of thymocytes treated with various death-inducing stimuli or in death-by-neglect of mature, periph eral T cells (Fig. 7g-i) .

To validate these findings, we crossed a different T cell- specific Cre mouse line, RORgammact Cre (Eberl, et al . Science 305, 248-51 (2004)), with Gchl (fl/fl) mice, generating Gchl;RORc mice. Again, loss of GCH1 in T cells did not affect thymocyte development or peripheral T cell homeostasis. Importantly, GCH1 was again shown to be indispensable for mature T cell prolifera tion (Fig. li) . B cell-specific deletion of Gchl using MBl-Cre (Hobeika, E. et al . Proc. Natl. Acad. Sci. 103, 13789-13794 (2006)) did not affect B cell development, proliferation or class switch recombination in response to the mitogenic stimulus LPS (Fig. 8) . These data show that GCH1 has no apparent effect on the development or homeostasis of T cells nor on the develop ment or activation of B cells. However, inactivation of Gchl markedly impairs the proliferation of mature, antigen receptor stimulated CD4⁺ and CD8⁺ T cells.

Example 3: Loss of GCH1 abrogates T cell-mediated autoimmune diseases

To investigate whether Gchl-ablated, BH4-deficent T cells are defective in vivo, we evaluated several models of T cell- dependent inflammation. First, we employed a T cell-dependent colitis model in which naive, CD4⁺ T cells are transferred into Ragl ^/ hosts (Sledzihska, A. et al . PLoS Biol. 11, el001674 (2013) ) . Because Ragl ^/ hosts lack regulatory T cells, the un- checked donor T cells activate and proliferate in response to the intestinal microflora, orchestrating an intestinal inflamma tory incursion that culminates in autoimmune colitis and severe intestinal damage¹². In contrast to control CD4⁺ T cells, the transfer of Gchl-deficient CD4⁺ T cells failed to elicit intesti nal damage and colitis (Fig. 2a) . Moreover, transfer of Gchl- ablated CD4⁺ T cells triggered a dramatically lower influx of im mune cells compared to control cell transfer, corresponding with less inflammation (Fig. 2b) .

Next, we utilized a model of allergic airway inflammatory disease in which immune cells, particularly CD4⁺ Th2 cells and eosinophils, are central to disease pathology. Allergic inflam mation and bronchial hyperresponsiveness were induced by inhaled OVA challenges (days 14-17) after an initial sensitization to the allergen, ovalbumin (OVA) (i.p. day 0 and 7) (Fig. 2c) . Com pared to controls, Gchl ;Lck mice showed significantly fewer CD45⁺ cells, eosinophils, and T cells in bronchoalveolar lavage fluids obtained on day 21, four days after the last inhaled OVA chal lenge (Fig. 2c) . Moreover, T cell dependent OVA responses were severely weakened during primary immunization as well as with re-challenge (Fig. 9) . Gchl ;Lck mice also showed a significantly reduced inflammatory response after re-challenge compared to controls in a T cell-mediated skin dermatitis model (Martin, S.

F. et al . J. Exp. Med. 205, 2151-2162 (2008) ) (Fig. 2d) . Finally, we evaluated experimental autoimmune encephalomyelitis (EAE) , a CD4⁺ T cell-mediated demyelinating autoimmune disease of the CNS that models multiple sclerosis (Rangachari, M. & Kuchroo, V.

Journal of Autoimmunity 45, 31-39 (2013); Rangachari, M. et al . Nat. Med. 18, 1394-1400 (2012)). Experimental autoimmune enceph alomyelitis (EAE) is an animal model for multiple sclerosis and is characterized by demyelination in the brain and the spinal cord, that can be induced in naive animals by transfer of CD4⁺ T cells from diseased animals. Thus, it is generally considered that EAE represents a T cell mediated autoimmune disease Once more, Gchl ;Lck mice demonstrated significantly fewer neurologi cal defects than control mice over the course of the experiment with a reduced overall EAE severity (Fig. 2e, f) . Together, these data show that genetic ablation of Gchl in T cells allevi ates T cell-mediated inflammatory intestinal, airway, skin and brain diseases. Example 4 : Pharmacological inhibition of BH4 synthesis ameliorates T cell mediated inflammation

Next, we investigated whether depleting BH4 pharmacological ly influences T cell function. Instead of inhibition of GCH1 (Nar, H. et al . Proc. Natl. Acad. Sci. U. S. A. 92, 12120-12125 (1995); Nar, H. et al . Structure 3, 459-466 (1995)) we utilized an inhibitor that targets sepiapterin reductase (SPR) , the ter minal enzyme in the de novo BH4 synthesis pathway (Fig. 3a) . We have previously shown that the SPR inhibitor, SPRi3, reduces BH4 levels in sensory neurons in pain models, and normalizes the as sociated pain hypersensitivity (Latremoliere, A. et al . , supra). Purified naive CD4⁺ T cells treated with SPRi3 showed lower BH4 levels compared to vehicle-treated cells following TCR stimula tion (Fig. 3a) . Similar to the genetic ablation of Gchl , SPRi3 treatment did not affect the survival of non-stimulated T cells or the induction of early activation markers after stimulation (Fig. 10a, b) . Importantly, SPRi3-treated, TCR-stimulated CD4⁺ and CD8⁺ cells displayed a similar defect in proliferation as Gchl-ablated T cells (Fig. 3b, Fig. 10c) . EdU-pulse labelling revealed that SPRi3-treated control cells and Gchl-deficient cells displayed a significantly lower percentage of S-phase cells 28 hours after TCR stimulation than vehicle-treated con trol cells (Fig. 3c, d) . The lack of S-phase entry eventually culminates in increased cell death of Gchl-null cells after TCR stimulation (Fig. 3e) . Thus, targeting a distal enzyme in the BH4 pathway phenocopies the T cell defects observed upon genetic ablation of Gchl.

We next assessed the efficacy of SPRi3 administration on the autoimmune colitis and allergic inflammatory models. Similar to T cell-specific knockout of Gchl, SPRi3 administration signifi cantly ameliorated colitis, and greatly diminished the intesti nal infiltration of T cells and other immune cells after CD4⁺ T cell transfer (Fig. 3f) . SPRi3 treatment also reduced the immune cell infiltration into the lung after aerosol OVA challenge (Fig. 3g) . Importantly, to translate these findings in mice to human T cells, we isolated human naive CD4⁺ cells from different healthy donors (n=4). Following anti-CD3/CD28 stimulation,

SPRi3-treated, freshly isolated CD4⁺ human T cells also exhibited significantly reduced proliferation compared to vehicle-treated cells (Fig. 3h) . Moreover, we saw a significant decrease in pro liferative capacity in SPRi3-treated human effector CD4⁺ T cells after anti-CD3/CD8 re-stimulation (Fig. 3i) . Taken together, these data show that the BH4 pathway is crucial for T cell pro liferation in mouse and human and, importantly, can be pharmaco logically blocked to abrogate autoinflammatory T cell responses in vivo.

Example 5 : BH4 can directly reduce Fe³⁺ and is critical for mitochondrial respiration in activated T cells

BH4 is an essential co-factor for several aromatic amino ac id hydroxylases which are required for the synthesis of seroto nin, epinephrine, norepinephrine, and dopamine (Werner, E. R., et al . Biochem. J. 438, 397-414 (2011)). We therefore first evaluated the levels of each of these, as well as their corre sponding amino acid precursors, in resting and activated T cells, and in the supernatant after TCR-stimulation . T cells from control and Gchl-mutated animals either showed no expres sion or no significant differences under the conditions tested (Fig. 11) . To gain insight into the molecular mechanism of BH4- dependent processes in activated T cells, we performed gene ex pression profiling in TCR-stimulated CD4⁺ T cells from control and Gchl ;Lck mice. Analysis of the significantly altered genes confirmed that loss of GCH1 did not apparently affect early T cell activation (The data are accessible through GEO Series ac cession number GSE108101). Intriguingly, detailed analysis un covered that several genes involved in iron homeostasis or the regulation of iron availability were upregulated. Upregulation of genes critically involved in iron metabolism were confirmed by Western blot of activated Gchl-ablated T cells (Fig. 4a), suggesting an involvement of BH4 in iron regulation upon T cell activation .

Ferritin binds and stores bio-inactive ferric iron (Fe³⁺) and releases it in a controlled manner as bioactive ferrous iron (Fe²⁺) . Haemoxygenase-I (HO-I) catalyzes the degradation of haeme, in turn releasing Fe²⁺. Mitoferrin is an iron transporter located at the mitochondrial membrane which transports Fe²⁺ into the mitochondria, and Frataxin is involved in the assembly of iron-sulfur (Fe-S) clusters, which are vital for the oxidation- reduction reactions of mitochondrial electron transport, in par- ticular for complex I and complex II of oxidative phosphoryla tion which have multiple Fe-S clusters. We suggested that BH4 might affect Fe²⁺ availability in cells. Indeed, we found an old and entirely ignored biochemical report that BH4 can reduce mo lecular ferric (Fe³⁺) iron to ferrous (Fe²⁺) iron. We confirmed using doses of BH4 that are physiologically present in activated T cells, expanding these results in that BH4 also efficiently reduces ferri-cytochrome C to ferro-cytochrome C (Fig. 4b) . Im portantly, total iron levels were significantly reduced in TCR- activated Gchl-ablated CD4⁺ T cells compared to control cells while unstimulated cells had similar iron content (Fig. 4c) .

These observations, combined with the fact that BH4 facili tates the reduction of ferric-cytochrome C to its ferrous form, suggested that BH4 deficiency may result in mitochondrial dys function. Supporting this, both Gchl-deficient (Fig. 4d) and SPRi3-treated (Fig. 4e) CD4⁺ T cells synthesized lower amounts of ATP than control cells as early as 10 hours after anti-CD3/CD28 stimulation. We next assessed the efficacy of the electron transport chain (ETC) system in activated T cells. After anti- CD3/CD28 stimulation, mitochondrial respiration was significant ly lower in both Gchl-ablated and SPRi-3-treated cells compared to control cells (Fig. 4f) , and this defect was due to decreased activity of the ETC complex I and, to a lower extent, complex II (Fig. 4g-i) . As a consequence of impaired OXPHOS, reactive oxy gen species (ROS) levels were significantly elevated in activat ed CD4⁺ T cells from Gchl-ablated animals and SPRi3-treated cells compared to controls (Fig. 4j, Extended Fig. 6a) . The superoxide scavenger, N-acetylcysteine (NAC) only partially rescued the proliferation defect of Gchl-ablated T cells (Fig. 4k), suggest ing that elevated ROS is not the only deleterious consequence of BH4 depletion and iron dysregulation . BH4 is also an essential co-factor for nitric oxide synthases (NOS) in the production of nitric oxide (NO) . It was reported recently that at low BH4 lev els, inducible NOS (iNOS) becomes uncoupled and generates super oxide at the expense of NO and thus may be the source of the el evated superoxide levels (Chen, W. et al . J. Biol. Chem. 286, 13846-13851 (2011)). However, under our experimental conditions we did not observe detectable iNOS expression nor NO production until several days after T cell activation (Fig. 13a-c) , whereas we observed elevated ROS levels, and mitochondrial dysfunction, after only 10 hours of stimulation paralleling GCH1 induction (see data above) . Overall, not excluding other possible mecha nisms, our data show that antigen receptor stimulated, BH4- depleted T cells display defective iron metabolism and mitochon drial dysfunction.

Example 6 : Enhanced BH4 production super-activates T cells

To investigate whether elevated GCH1 enhances T cell func tion in vivo, we crossed the Lck-Cre driver line to Cre- recombinase dependent, GCH1 over-expressing mice (hereafter re ferred to as GOE) , generating GOE;Lck mice that express an HA- tagged human GCH1 transgene only in T cells. The GOE;Lck mice expressed GCH1-HA in naive CD4⁺ T cells, observed by Western blot using antibodies directed against GCH1 or the HA tag (Fig. 5a) .

T cell development and homeostasis of peripheral CD4⁺ and CD8⁺ T cells were unaffected in these mice, though in the periphery there was a significant increase in the proportion of effector T cells (Fig. 14a-c) . Anti-CD3/CD28-stimulated CD4⁺ T cells from the GOE;Lck mice had elevated BH4 levels compared to controls (Fig. 5b) and displayed enhanced proliferation upon activation (Fig. 5c) . Of note, we only observe this enhanced proliferation following antigen receptor/CD28 stimulation; GCH1 overexpression per se does not result in proliferation of unstimulated naive T cells. To validate that elevated GCH1 increases proliferation of activated T cells, we used additional T cell-specific Cre lines to drive GCH1-HA expression. We crossed the GOE mice to CD4-Cre mice, and observed a similar increased proliferation of activat ed T cells (Fig. 5d) , as well as greatly enhanced IL-2 and IFNy secretion (Fig. 5e) , and increased effector T cell populations in the periphery (Fig. 14d, e) . These effects were further con firmed in a third genetic model by stimulating CD4⁺ T cells from GOE;ERT-Cre mice in the presence of 4-hydroxytamoxifen to induce GCH1-HA expression in vitro (Fig. 15a-c) . These data show that genetic over-expression of the BH4 pathway has the opposite ef fect of depleting GCH1/BH4, leading to enhanced T cell prolifer ation and function.

We next asked whether pharmacological elevation of BH4 en hanced T cell function. Sepiapterin (SP) is a metabolite of the BH4 salvage pathway distinct from the GCHl-dependent de novo synthesis of BH4 but is enzymatically converted to BH4 through the SPR enzyme (Fig. 5f, Fig. 15d) . As expected, SP-treated, an- ti-CD3/CD28-stimulated CD4⁺ T cells displayed elevated levels of BH4 (Fig. 5f) . This increased BH4 production was accompanied by enhanced proliferation of stimulated control CD4⁺ and CD8⁺ T cells (Fig. 5g) . Entry to S-phase after T cell activation was significantly boosted in SP-treated T cells (Fig. 15e, f) . Fur thermore, treatment of stimulated CD4⁺ T control cells with BH4 itself dramatically increased both proliferation and IL-2 secre tion (Fig. 5h) . Indeed, we could rescue the proliferative and S- phase entry defects observed in Gchl-ablated T cells by treating them with either SP (Fig. 5g, Fig. 15e, f) or addition of BH4 (Fig. 15g) . ROS levels in stimulated T cells overexpressing GCH1 or treated with SP were lower than in control cells and similar to unstimulated cells (Fig. 5i) and stimulated CD4⁺ T cells treated with SP produced significantly more ATP (Extended Fig.

9h) . Thus, genetic and pharmacologic induction of BH4 markedly enhances T cell proliferation and function.

Example 7 : BH4 enhances anti-cancer immunity

Drugs that target T cell activation checkpoints, such as an- ti-PD-Ll and anti-CTLA-4, offer a new hope to cancer patients (Sharma, P., et al . Nat. Rev. Cancer 11, 805-12 (2011)). Addi tional targets regulating T cell activation and function would be beneficial, as combination therapy shows the most promise for successful cancer immunotherapy treatment. Therefore, we asked whether hyperactivation of the GCH1/BH4 pathway in T cells might promote anti-cancer immunity. We orthotopically injected E0771 breast cancer cells into syngeneic mice to generate mammary tu mors, which were then monitored over several weeks. GOE CD4 mice, unlike controls, completely rejected tumor growth (Fig.

6a) . Moreover, therapeutic treatment of mice carrying estab lished E0771-derived mammary tumors with BH4 slowed the growth of the tumors significantly (Fig. 6b) . The BH4-treated tumors displayed increased frequencies of activated effector CD4⁺ and CD8⁺ cells among the infiltrating T cell population compared to vehicle-treated tumors (Fig. 6c), confirming that BH4 admin istration increased T cell activation and enhanced their anti tumor response.

Given the anti-tumor activity of GCH1/BH4 in T cells we hy pothesized that cancer cells might have evolved a way to dampen this pathway to benefit their survival. The essential amino acid tryptophan is catabolized in tumor tissues by indoleamine-2 , 3- dioxygenase (IDO), creating an immunosuppressive environment in which tryptophan reduction is thought to induce T cell anergy.

It has been proposed that IDO-produced kynurenine metabolites directly induce immunosuppression via increased transdifferenti ation of CD4⁺ T cells into Tregs, as well as via activation of the aryl hydrocarbon receptor (AhR) on dendritic cells and mac rophages to induce Treg differentiation (Mezrich, J. D. et al .

J. Immunol. 185, 3190-3198 (2010)). It has also been suggested that kynurenine can directly affect T cell proliferation (Ter- ness, P. et al . J. Exp. Med. 196, 447-457 (2002)). Intriguingly, in a recent chemical screen to identify modulators of the BH4 pathway, we found that the kynurenine metabolite xanthurenic ac id also inhibits SPR function and thus blocks BH4 production. Indeed, we found that addition of kynurenine to T cell cultures did significantly reduce T cell proliferation (Fig. 6d) and, im portantly, the proliferative capacity was fully restored by ad dition of BH4 (Fig. 6e) . In addition, in activated T cells, BH4 treatment rescued the elevated ROS levels observed upon

kynurenine treatment (Fig. 6f) . We have therefore demonstrated that kynurenine-mediated suppression of T cells is through inhi bition of the BH4 synthetic pathway. Thus, BH4 enhances anti tumor activity of T cells and counteracts kynurenine-dependent immunosuppressive effects.

Example 8: Further BH4 biological activity antagonists inhibit T cell activity

Fluphenazine hydrochloride and EGFR III Inhibitor were pur chased from Sigma (BP167) and Millipore (US1324833) , respective ly. For in vitro use, both were dissolved in DMSO to a stock concentration of lOmM.

Experiments on sciatic nerve injury were performed as previ ously described (Decosterd, C.J. Woolf. Pain, 87 (2000), pp .

149-158) . After 3 days the injured nerve was extracted, and pro teins extracted in RIPA buffer and proceeded to western blot ting .

For lymphocyte proliferation, indicated T cells were puri fied from spleens and lymph nodes of mice using microbeads (CD4⁺; Miltenyi Biotec) . Purified cells were stained with Cell Trace Proliferation Kit ( Invitrogen) . 96 U-shaped plates were coated with anti-CD3 (Biolegend) with/without anti-CD28 (Biolegend) at the indicated concentrations in PBS for 3 hours at 37°C. T cells were plated at 10⁵ cells/well in IMDM+PenStrep+Lgly+10% FCS . Al ternatively, the cells were cultured for 24 hours and expression of activation markers (CD62L, CD25) were analyzed using Flow Cy tometry or the cells were cultured for 3 days and proliferation was assayed by flow cytometry on viable cells (DAPI-negative) .

Protein blotting was carried out using standard protocols. Blots were blocked for 1 h with 5% BSA in TBST (lx TBS and 0.1% Tween-20) and were then incubated overnight at 4 °C with primary antibodies, diluted in 5% BSA in TBST (1:1,000 dilution) . Blots were washed three times in TBST for 15 min and were then incu bated with HRP-conj ugated secondary antibody (1:2,500 dilution;

GE Healthcare, NA9340V) for 45 min at room temperature, washed three times in TBST for 15 min and visualized using enhanced chemiluminescence (ECL Plus, Pierce, 1896327).

The results in figures 16-18 show that both fluphenazine hy drochloride and EGFR III Inhibitor inhibit T-cell activity via the BH4 pathway.

Discussion

T cells play an essential role in combatting invading patho gens as well as providing anti-cancer immunity. Conversely, self-reactive T cells can cause devastation manifesting in auto immune diseases. Emerging data highlight the intimate relation ship between T cell function and cellular metabolism. Identify ing pathways that coordinate metabolic processes with inflamma tory effector functions is of paramount therapeutic importance to not only enhance T cell function in the case of cancer immu notherapy, but, equally crucial, to repress their function under conditions of autoimmunity. Here, we have identified that the BH4 pathways, GCH1 and it downstream metabolite BH4, is engaged in activated T cells. Genetic inactivation of Gchl with multiple T cell-specific Cre lines, as well as pharmacological inhibition of BH4 production, leads to markedly reduced CD4⁺ as well as CD8⁺ T cell proliferation via disruption of iron homeostasis and mi tochondrial dysfunction. Furthermore, over-production of BH4, results in T cell hyper-proliferation.

Genetic or pharmacologic inhibition of the BH4 metabolic pathway markedly abrogated the severity of autoimmunity and al lergies in several different T cell-mediated model systems. Con versely, T cell specific overexpression of Gchl and administra tion of BH4 markedly enhanced anti-cancer immunity in an ortho- topic breast cancer model, supporting the notion that the BH4 pathway is an integral checkpoint control in T cell prolifera tion. The tryptophan metabolite kynurenine, generated by the en zyme IDO, suppresses immunity in cancer as well as locally in the intestine. Kynurenine metabolites inhibit SPR and hence BH4 production (Haruki, H., et al . J. Biol. Chem. 291, 652-657

(2016)) . Our finding that BH4 is a key metabolite for T cell proliferation therefore suggests a novel, tryptophan-dependent immunosuppressive pathway mediated by kynurenine metabolism. In deed, we showed that kynurenine inhibits T cell proliferation in isolated T cell cultures, and that that this can be rescued by BH4. These data suggest that BH4 administration could be an ef fective therapy to both bolster T cell proliferative responses as well as bypass specific endogenous tumour immunosuppression mechanisms. Mechanistically, we show that BH4 plays an integral role in regulating iron homeostasis during T cell activation and that Gchl-ablated T cells have lower iron content after activa tion. BH4 can directly reduce ferric iron to ferrous iron, in cluding reduction of cytochome-c-Fe³⁺ to cytochrome-c-Fe²⁺, af fecting electron transport via complex I and II and consequently mitochondrial respiration, resulting in enhanced ROS production and impaired ATP required to drive T cell energetics and prolif eration. Additional effects of BH4 in ROS scavenging or minute NO changes, even though we did not detect such changes at time points where BH4 is already required, cannot be excluded. Im portantly, it has been known that inhibiting iron uptake by blocking the transferrin receptor, which is induced on activated T cells, impairs cell cycle progression of T lymphocytes. Moreo ver, several studies in animals and humans have shown that nu tritional iron deficiency is associated with impaired T cell proliferation and delayed-type hypersensitivity responses while humoral immunity appears to be largely preserved. Moreover, iron-deficiency anemia has been associated with increased cancer incidence. Thus, we have uncovered a key molecular link between iron metabolism, mitochondrial bioenergetics and T cell prolif eration .

In conclusion, we have uncovered that BH4 is an essential metabolite required for effective mature T cell proliferation in vitro and in vivo. Furthermore, we find that BH4 depletion ap pears to be a link between the local immunosuppressive tumor en- vironment and reduced T cell function. Induction of GCH1 and BH4 overcomes such inhibition to enhance immunity and inhibit tumor growth. Since BH4 acts in a similar way in human T cells, block ade of the BH4 pathway could be a viable option to abrogate pro- inflammatory auto-aggressive T cells under pathological disease conditions, whereas supplementation of this metabolite could be a novel way to enhance anti-tumor immunity.

Claims

Claims :

1. The method of inhibiting T cell activity comprising inhibit- ing BH4 biological activity in said T cell.

2. The method of claim 1 wherein said T cell is a CD4⁺ or CD8⁺ T cell.

3. The method of claim 1 or 2, wherein said T cells are from a patient suffering from a T cell-mediated autoimmune disease or T cell-mediated hypersensitivity.

4. The method of any one of claims 1 to 3, wherein said T cell- dependent autoimmune disease or T cell-dependent hypersensitivi ty is selected from multiple sclerosis, allergic contact derma titis, type 1 diabetes mellitus, rheumatoid arthritis, giant cell arteritis, reactive arthritis, coeliac disease, Rasmussen's encephalitis, acute disseminated encephalomyelitis, Sjogren's syndrome, allergic granulomatosis, including Churg-Strauss syn drome, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's Disease, sarcoidosis, Wegen er's granulomatosis, autoimmune encephalomyelitis, oophoritis, microscopic colitis, uveitis, primary biliary cirrhosis, autoim mune hepatitis, ankylosing spondylitis, contact dermatitis, atopic dermatitis, chronic thyroiditis, a T cell-mediated aller gy, a T cell-mediated food allergy, T cell-mediated allergic contact dermatitis, graft versus host disease, transfusion- associated graft versus host disease, Heiner syndrome, T cell- mediated hypersensitivity reactions to drugs, T cell-mediated skin inflammation.

5. The method of any one of claims 1 to 4, comprising treating the T cell with an BH4 biological activity antagonist.

6. The method of any one of claims 3 to 5, comprising diagnos ing a patient with a T cell-mediated autoimmune disease or T cell-mediated hyper-sensitivity or a predisposition thereto and then treating the patient with a BH4 biological activity antago nist.

7. The method of any one of claims 1 to 6, comprising wherein said T cells are sensitized against an auto-antigen or a micro flora antigen, preferably wherein the method comprises detecting said sensitized T cells in a patient and/or obtaining said sen sitized T cells from the patient.

8. The method of any one of claims 5 to 7, wherein the BH4 bio logical activity antagonist is selected from an sepiapterin re ductase inhibitor, GTP cyclohydrolase 1 inhibitor, protein tyro sine phosphatase inhibitor, aldo-keto reductase family member C3 inhibitor, aldo-keto reductase family member BIO inhibitor, di hydrofolate reductase inhibitor, pterin-4-alpha-carbinolamine dehydratase inhibitor, dihydropterine reductase inhibitor, or combinations thereof.

9. The method of any one of claims 5 to 8, wherein the BH4 bio logical activity antagonist is selected from

a GTP cyclohydrolase I inhibitor selected from a substituted py rimidine, preferably hydroxyl, amino or halogen substituted py rimidine, in particular preferred 2, 4-diamino-6- hydroxypyrimidine, 2, 5-diamino-6-hydroxypyrimidine, 4, 5-diamino- 6-hydroxypyrimidine, 4 , 5-diaminopyrimidine, and 4 , 6-diamino-2- hydroxypyrimidine ; an oxidized pterin, preferably neopterin, xanthopterin, isoxanthopterin and biopterin; a reduced pterin, preferably 7, 8-dihydro-D-neopterin, (6R, S) -5, 6, 7, 8-tetrahydro-D- neopterin, 7 , 8-dihydrofolic acid and 5, 6, 7, 8-tetrahydrofolic ac id;

a GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula (I) :

prodrug, or pharmaceutically acceptable salt thereof, wherein:

R¹ _» R², R³, and R⁴ are each, iide; adonily, H_» optionally substituted alkyl, or R¹ and R³, R³ and R³ _» or R² and R⁴ combine to form a double bond,

R⁵ _» R⁶, and R⁷ are each, indepndentlj , H or optionally substituted Cn alkyl, and

wherein one and only one of R¹ and R² _» R² and R³, or R² and R⁴ combine to form a double bond, and

when R⁵ _»

and R' are H, R¹ and ² combine to form a double bond and R^J is H, or when I^s, R⁶ _» and R⁷ are H, R² and l¹ combine to form a double bond and R¹ is H, R⁴ is not -C¾ H_S, ~a¾r-€*ί €H)_; -CH^p-C^HrCHs), %CH=€¾, -

CH₂C(=OKp- H4-OMe), -CH₂CC ))NH-(o-C6¾-OEt), -Cll₂C(=0)NH- 2- methoxy-S-chloro-C^), -CJ¾C(-0)NH-{2-iiietliylcydohexy 1), or -CHiCC^OJN H-

(p-C^R_t'SOsfazepane)) ;

a GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula (II-A) or Formula (II-B) :

or a tautomer, prodrug, or pharmaceutically acceptable salt thereof, wherein R¹, R² and R³ are each, independently, H or op tionally substituted Ci_ ₆ alkyl; a GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula (III) :

(ill), or a tautomer, prodrug, or pharmaceutically acceptable salt thereof, wherein

X¹ is O or NR¹;

X² is O or NR²;

R¹ and R² arc each, independently, selected from II, or optionally substituted

Ci (, alkyl;

R^J is H, halogen, or NR*R⁹, or R³ combines with R⁴ to form an oxo group; and

R⁴ combines with R¹ or R² to form a C=N bond or R⁴ combines with R^J to form an oxo group;

R⁵, ⁶, R¹, R\ end K^¾ are each, independently, H or optionally substituted C_|4 alkyl; anil

when R\ R’’. and R^† are I I. X'is NR¹. R¹ and R¹ combine to form ;t ( '-N

double bond and X is M 1, Rs is not 1 1 or M fo and

when R'. K^h, and R ' are H, X is NH„ R' combines with R ^* to form an ox

group and X is NR², R² is not H; a GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula (IV-A) or Formula (IV-B) :

tautomer, prodrug, or pharmaceutically acceptable salt thereof; or according to

tautomer, prodrug, or pharmaceutically acceptable salt thereof, wherein R¹ , R², R³, R^s, R⁶, and R⁷ are each, independently, H or optionally substituted: C_t-& alkyl· a GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula (V-A) or Formula (V-B) :

tautomer, prodrug, or pharmaceutically

acceptable salt thereof,

tautomer, prodrug, or pharmaceutically acceptable salt thereof, wherein each of R¹, R⁶, and R⁷, Is H or optionally substituted

C | .6 alkyl; a GTP cyclohydrolase I inhibitor selected from a compound having a structure according to Formula (VI) :

tautomer., prodrug, or pharmaceutically

acceptable sab thereof, wherein each of R¹, ², R⁶, and R⁷ is, independently, H or optionally substituted t'n alkyl;

a protein tyrosine phosphatase inhibitor selected from etidro nate, -Bromo-4-hydroxyacetophenone, 4- (Bromoacetyl) anisole; sepiapterin reductase inhibitor selected from N-acetylserotonin, N-acetyldopamine, N-acetyl-m-tyramine, N-chloroacetyldopamine, N-chloroacetylserotonin, N-methoxyacetyldopamine and N- methoxyacetylserotonin;

a sepiapterin reductase inhibitor selected from a structure of Formula (VI I ) ,

wherein

each of X‘ and X is. independentl , N, ( -il, otf ( -halogen;

A is a single bund. C(— O i. or SO;:

R¹ i ( Cl k) OR \ halogen amino. CN, SOJR X MISO di ' \ M l IC( O JR ¹ \ or C(- 0)N(R^,A)₂;

each R^iA is, independently, H or optionally substituted i Vr, alky l; n is 0, 1 , or 2;

R^~ is i ikOUd opiiunuily subsiiiute C _{E -r>} alkyl, optionally substituted k ;_,_< cycloalkyl. optionally substituted ary l. optionally substituted heteroeye lyl. or optionally substituted heterouryh

R ^'" k 1 1 or optionalh substituted C _{s . r} alkyl;

R '^A and R’¹⁵ are both I I, or ^,A and R^1B combine to fomt— C);

R ^IA and R ^B are both 11, or R ^IL and R¹ combine to form =0;

each ofR^' and R^:' is. independent;) . I I. optional!) substituted Ci.- alky I. optionally substituted Ch.io cycloalkyl optionally substituted alkaryl, or optionally substituted alkheteroaryl; and

wliei cin vv neii

are each 11, and R is 1 1. R' k not 1 1 ;

SPRi3, sulfasalazine or a sulfa analogon of sulfasalazine;

a sepiapterin reductase inhibitor selected from a kynurenine me tabolite, preferably xanthurenic acid, N-acetylserotonin, kynurenic acid, 8-hydroxyquinaldic acid, picolinic acid, 3- hydroxyanthranilic acid;

a dihydrofolate reductase inhibitor selected from methotrexate, aminopterin, 10-propargyl-5, 8-dideazafolate; 2 , 4-diamino, 5- ( 3 ' , 4 ' -dichlorophenyl ) , 6-methylpyrimidine ; trimetrexate ; py rimethamine; trimethoprim; pyritrexim 5,10- dideazatetrahydrofdate ; 10-ethyl, 10-deazaaminopterin; or py- rimethamine ;

an inhibitory nucleic acid against sepiapterin reductase, GTP cyclohydrolase 1, protein tyrosine phosphatase, aldo-keto reduc tase family member C3, aldo-keto reductase family member BIO, dihydrofolate reductase, pter-in-4-alpha-carbinolamine dehydra tase, dihydropterine reductase;

an antibody against sepiapterin reductase, GTP cyclohydrolase 1, protein tyrosine phosphatase, aldo-keto reductase family member C3, aldo-keto reductase family member BIO, dihydrofolate reduc tase, pter-in-4-alpha-carbinolamine dehydratase, dihydropterine reductase .

10. The method of any one of claims 1 to 9, wherein the T cells are from a patient who is not suffering from pain, neurotrans mitter dysregulation, nitric oxide dysregulation, a non-T cell- mediated inflammation, allograft rejection related to nitric ox ide production, inflammation caused by induced nitric oxide in immune cells.

11. The method of any one of claims 1 to 10, wherein inhibiting BH4 biological activity in said T cell is in vitro and/or ex vi vo, preferably of isolated and/or purified T cells.

12. The method of claim 11, further comprising introducing or reintroducing the treated T cell into a patient.

13. A BH4 biological activity antagonist for use in the treat ment of a T cell-mediated autoimmune disease or T cell-mediated hypersensitivity, preferably wherein said treatment comprises a method of any one of claims 1 to 12.

14. SPRi3 for use in the treatment in the treatment of colitis, asthma, psoriasis or multiple sclerosis, preferably wherein the patient has received T cells.

15. Kit-of-parts suitable in a use according to any one of claims 1 to 12 comprising (i) a BH4 biological antagonist and (ii) a T cell culturing medium and/or a T cell adsorbent and/or a T cell-specific drug delivery agent.