WO2019175328A1 - Bh4pathwayactivationandusethereoffortreatingcancer - Google Patents

Abstract

The present invention relates to a method of increasing T cell activity comprising increasing BH4 biological activity in said T cell, ex vivo and in vivo, in particular in the treatment of cancer, as the invention enhances T cell proliferation and anti- tumour immunity. The invention further relates to kits for such methods.

Description

BH4 PATHWAY ACTIVATION AND USE THEREOF FOR TREATING CANCER

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 enzymes, including nitric oxide synthases, aromatic amino acid hydroxylases (phenylalanine, tyrosine and tryptophan hydroxylases) and the alkylglycerol mono-oxygenase. Through these enzymes, BH4 is required to produce nitric oxide (NO) , catabolize phenylalanine, 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 typically is the rate-limiting enzyme for BH4 biosynthesis) , FTPS (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 activators or agonists to these enzymes exist for positive modulation, also referred to as increasing BH4 biological activity.

Rabender et al • / Cancer Research 2016, DOI : 10.1158/1538- 7445.AM2016-3383, proposes that sepiapterin, being a precursor of BH4, could potentially normalize tumor vasculature as a result of reduced levels of nitric oxide.

US 4,752,573 discloses the use of pterins, such as BH4, for increasing the activity of lymphokines such as in combination with IL-2. Kurupati et al Oncoscience 5.1-2 (2018): 1, discloses the use of fenofibrate for stimulating tumor antigen specific CD8⁺ T cells in order to promote them switching from glucose to fatty acid catabolism and prevent them from starving within the tumor microenvironment .

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 conca- navalin A-induced lymphocyte activation.

So far unrelated to the BH4 pathway, cancer is one of the leading causes of morbidity and mortality worldwide, despite recent progress in targeted therapies. Drugs that target T cell activation checkpoints ("immune checkpoint inhibitors") , such as anti-PD-Ll and anti-CTLA-4, offer a new hope to cancer patients

(see e.g. Sharma, P • / Wagner, K • / Wolchok, J. D. & Allison, J. P. Novel cancer immunotherapy agents with survival benefit: re- cent successes and next steps. Nat. Rev. Cancer 11, 805-12

(2011) ; Curran, M. A • / Montalvo, W • / Yagita, H. & Allison, J. P. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc. Natl. Acad. Sci. 107, 4275-4280 (2010) and

Pedicord, V. a, Montalvo, W • / Leiner, I. M. & Allison, J. P. Single dose of anti-CTLA-4 enhances CD8+ T-cell memory formation, function, and maintenance. Proc. Natl. Acad. Sci. U. S. A. 108, 266-71 (2011)). However, known immune checkpoint inhibitors are neither effective in all patients nor in all cancers.

Accordingly, there remains a goal to find further immune checkpoint inhibitors in order to improve cancer therapy.

Summary of the invention

The present invention is based on the discovery that T-cell specific GCH1 overexpression in mice as well as therapeutic administration of BH4 to mice enhances anti-tumour immunity (see in particular examples 6 and 7) .

In a first aspect the invention provides a method of increasing T cell activity (such as IL-2 secretion, IFN-gamma secretion and/or proliferation) comprising increasing BH4 biological activity in said T cell, especially in or for the prevention or treatment of a cancer. Said T cells can be ex vivo (in vitro) or in vivo. Accordingly, the invention further relates to a method of preventing or treating a cancer comprising increasing BH4 biological activity in said T cell (s) . Related thereto, the invention provides a BH4 biological activity agonist for use in the prevention or treatment of a cancer. Also related thereto, the invention provides a method of manufacturing a pharmaceutical composition that is capable of increasing BH4 biological activity or that comprises a BH4 biological activity agonist for use in a prevention or treatment of a cancer. In particular, the invention provides sapropterin, sepiapterin, or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of a cancer; and related thereto is provided a method of manufacturing a pharmaceutical composition comprising sapropterin, sepiapterin, or a pharmaceutically acceptable salt thereof for use in preventing or treating a cancer.

The invention further relates to a kit-of-parts comprising (i) a BH4 biological agonist and (ii) a T cell culturing medium and/or a T cell adsorbent and/or a T cell-specific drug delivery 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 detailed descriptions of e.g. BH4 biological activity or agonists, relate to preferred embodiments of all aspects of the invention.

Detailed description of the invention

The present invention relates to a method of increasing T cell activity comprising increasing BH4 biological activity in said T cell. Such a method can be performed ex vivo and in vivo, in particular in the prevention or treatment of a cancer, as the invention enhances anti-cancer immunity.

The inventive treatment of T cells can be specific to T cells or systemic (in vivo) . Specificity however is preferred.

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

(2007) 32:751-756).

The present invention also relates to a method for increasing the immunoreactivity of T cells, comprising increasing the BH4 biological activity of the cells. This can result in in- creased immunoreactivity of the cells to an antigen. Such an antigen is preferably a tumour antigen (tumour-specific or tumour- associated) , such as HER2/neu, EGFR, VEGF, CAMPATH1, CD22, CA- 125, HLA-DR, MUCIN-1, Survivin, Alpha-1 fetoprotein, tyrosinase, PSMA, PSA, MAGE-A1, MAGE-A1, and NY-ESO-1. It can also be a tumour antigen as disclosed in Coulie et al. (Coulie, Pierre G • / et al. "Tumour antigens recognized by T lymphocytes: at the core of cancer immunotherapy." Nature Reviews Cancer 14.2 (2014) : 135.) and Scott et al. (Scott, Andrew M • / Jedd D. Wolchok, and Lloyd J. Old. "Antibody therapy of cancer." Nature Reviews Cancer 12.4 (2012): 278.) in particular Table 2 thereof, all incorporated herein by reference. Preferably, within the context of the present invention, the T cells are sensitized against (or reactive to) a tumour antigen, such as the ones just mentioned.

The T cells can be from a patient having or predisposed of developing a cancer. 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 composition, or by treating the T cells ex vivo (e.g. after isolation from the patient or a healthy individual) and reintroduction of the T cells into the patient. According to the therapeutic aspect, the invention provides a BH4 biological activity agonist for use in the prevention or treatment of a cancer.

In the context of the invention, preferred cancers are breast cancer, lung cancer, ovarian cancer, cervical cancer, hepatocellular carcinoma, renal cell carcinoma, thyroid cancer, neuroendocrine cancer, gastro-oesophageal cancer, bladder cancer, colorectal cancer, glioma, prostate cancer, pancreatic cancer, stomach cancer, oesophagal cancer, liver cancer, melanoma, glioblastoma, leukemia and lymphoma.

According to preferred embodiments, the cancer is cancer which has relapsed at least once, preferably at least twice, more preferably at least three times. Preferably, relapse is defined as fulfilling the criteria for "progressive disease" according to version 1.1 of the RECIST guidelines (Eisenhauer, et al. "New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)." European journal of cancer 45.2 (2009) : 228-247• / in particular item 4.3.1 thereof) , after the criteria for "complete response" or "partial response" (especially the former) had initially been fulfilled according to said guidelines (see Eisenhauer et al • / 2009, in particular item 4.3.1 thereof) upon (e.g. at least 2 weeks, preferably at least 4 weeks, more preferably at least 8 weeks, even more preferably at least 12 weeks, especially at least 16 weeks or even at least 24 weeks) cancer therapy.

Herein, the terms "being predisposed of developing cancer", "predisposition to cancer" and the like shall mean that the patient has a risk of developing cancer which is higher than that of an age-matched and sex-matched control population. In particular, the terms shall mean that the patient has genetic risk factors for developing cancer, such as loss-of-function mutations in the BRCA1, BRCA2, BRCA3 or RADSID genes and/or other tumour suppressor genes.

According to preferred embodiments, the inventive therapy is combined with administration of a chemotherapeutic agent to the patient. The chemotherapeutic agent may be selected from the group of alkylating agents, platin-based chemotherapeutic agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors and cytotoxic antibiotics. Preferably, the chemotherapeutic agent is selected from the group of 5-fluorouracil, cis- platin, carboplatin, cyclophosphamide, doxorubicin, epirubicin, gemcitabine, taxanes such as paclitaxel or docetaxel, methotrexate, pemetrexed, vinorelbine and etoposide.

According to a particularly preferred embodiment, the inventive cancer prevention or treatment is in combination with a therapy with an immune checkpoint inhibitor (which may e.g. be administered in the same composition together the BCH4 activity agonist or in a different composition) . Immune checkpoint inhibitors are for instance discussed in La-Beck et al. (La-Beck, Ninh M. , et al. "Immune checkpoint inhibitors: new insights and current place in cancer therapy." Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy 35.10 (2015): 963-976.) and Topalian et al. (Topalian, Suzanne L • / Charles G. Drake, and Drew M. Pardoll. "Immune checkpoint blockade: a common denominator approach to cancer therapy." Cancer cell 27.4 (2015): 450- 461.), both of which are incorporated herein by reference. Immune checkpoint inhibitors are also disclosed e.g. in US 2017/0065716 Al_f US 2016/0311903 Al, US 2017/0037132 Al, and US 2013/0309250A1.

In preferred embodiments, the immune checkpoint inhibitor is a monoclonal antibody. Alternatively, or in addition thereto, the immune checkpoint inhibitor may be an inhibitor of PD-1 (such as nivolumab, pembrolizumab and pidilizumab) , PD-L1 (such as MPDL3280A, MEDI4736, and avelumab) , CTLA-4 (such as ipili- mumab, and tremelimumab) , IDO-1 (such as elotuzumab, INCB024360 and indoximod) , KIR (such as lirilumab) or LAG-3 (such as IMP321 and BMS-986016) .

As used herein, "prevention" should not be interpreted as an absolute success in the sense that a patient can never develop 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 prophylactic treatment is to be understood in the sense of a reduction of the risk of development not as a total risk avoidance. E.g. T cells can be treated even before clinical symptoms of the disease occur. Such T cells may be detected or isolated from the patient before a treatment.

The step of increasing 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 activity agonist, having the benefit that in vivo - apart from the T cells - the BH4 biological activity can remain unchanged - or can even be reduced.

The patient or subject to be treated and/or from whom the T cells are derived or obtained, may be a mammal, preferably a human. The patient preferably has or is predisposed to cancer, in particular any one of the cancers as mentioned herein. In preferred embodiments, 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, especially within the last months or even with the last two weeks or even within the last week) , in particular recombinant T cells such as CAR-T cells or TCR-T cells and/or who has recombinant T cells such as CAR-T cells or TCR-T cells (in the body) . Typically, the patient is in need of the inventive treatment. Preferably the patient does not have and/or is not predisposed to a condition selected from neurological conditions, psychiatric conditions, such as depression, and metabolic conditions, such as phenylketonuria and tetrahydrobiopterin deficiency. 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 background 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, in this aspect, related to the new mechanism 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 ailments 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 proliferate T cells as known in the art can be used.

Preferably, the T cells are not treated with IL-2 during or before the inventive method is applied.

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

As has been shown in the examples, such T cells, in particular activated T cells, can be super-activated (see in particular examples 6 and 7) . T cell precursors or immature T cells, such as DN3 thymocytes are typically not significantly affected by the inventive treatment, which reduces unwanted side-effects of the inventive treatment. The inventive BH4 biological activity increase is typically specific to peripheral T cells, with regard to T cells and their precursors in general.

In further preferred embodiments, said T cell is a recombinant T cell, i.e. a T cell whose genetic material has been altered using genetic engineering techniques (in vivo or in vitro/ex vivo, preferably the latter) , in particular for cancer therapy. The T cell may be a chimeric antigen receptor (CAR) T cell or a T cell with a recombinant T cell receptor (TCR) , the latter of which is also called "TCR-T cell" or "TCR-modified T cell" or "TCR-engineered T cell" in the field. The present invention further improves the efficacy of CAR-T cells and TCR-T cells. These cells may e.g. be treated with the BH4 biological activity agonist disclosed herein, either in vivo (e.g. when they have been reintroduced into the patient) or in vitro / ex vivo (e.g. before they are introduced into the cancer patient) .

CAR-T cells are T cells which are genetically manipulated in order to express a receptor (such as a monoclonal antibody or fragment thereof) which recognizes tumour cells. An overview is given in Barret et al. (Barrett, David M • / et al. "Chimeric an- tigen receptor therapy for cancer." Annual review of medicine 65 (2014) : 333-347.), incorporated herein by reference. FDA- approved CAR-T cancer therapies are e.g. axicabtagene ciloleucel and tisagenlecleucel . Further methods related to CAR-T cells are disclosed e.g. in US 2018/0037630 Al, US 2013/0287748 Al, US 2014/0227237 Al, WO 2015/142675 A2 and WO 2015/090230 Al, all of which are incorporated herein by reference.

TCR-T cells are T cells which are genetically manipulated in order to express a modified TCR which recognizes for a tumour antigen. TCR-T cells are further discussed e.g. in Karpanen et al. (Karpanen, Terhi, and Johanna Olweus. "T-cell receptor gene therapy—ready to go viral?." Molecular oncology 9.10 (2015):

2019-2042.) which is incorporated herein by reference.

According to the invention, increasing BH4 biological activity 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 patient 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 lymphocytes (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 surface markers, that can be used for binding a cell to a ligand of such surface markers, like CD4 or CDS . The surface markers may be found on the T cells of interest, and are preferably are specific to the T cells of interest, i.e. only minor amounts are found on other cells such that purification to the desired quantity (% as above) is achieved. An adsorbent, herein also referred to as T cell adsorbent, may be used to this effect. The adsorbent may be on a solid surface to facilitate ease of purification, 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 cancer or its prevention. This enhances anti-cancer immunity of the patient.

The invention also comprises diagnosing a patient with a cancer or a predisposition thereto and then treating the patient with a BH4 biological activity increase, e.g. with an agonist, in vivo or ex vivo (i.e. based on (re) introduction of treated T cells) - or in other words, detecting a cancer or a predisposition thereto in a patient and treating the patient with a BH4 biological activity increase, e.g. using the agonist, in vivo or ex vivo (i.e. based on (re) introduction of treated T cells). The diagnosis or detection is not necessarily performed together with the inventive treatment. The invention also relates to treating patients or T cells that have been diagnosed or detected. Said diagnosis or detection can e.g. a biopsy of a suspected tumour and subsequent histological analysis or a genetic test for cancer predisposition (e.g. a BRCA1 or BRCA2 test) . Such T cells may be sensitized (or reactive) against a tumour antigen as described above. 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 sensitive

against (or reactive to) a tumour antigen (such as described hereinabove) , 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 biological activity can be increased by using a BH4 biological activity agonist. The invention encompasses treating the T cell with an BH4 biological activity agonist. Such an antagonist can be an activator of any one of the enzymes in the synthesis of BH4. In particular, the BH4 biological activity agonist can be selected from an sepiapterin reductase activator, GTP cyclohydrolase 1 activator, protein tyrosine phosphatase activator, aldo-keto reductase family member C3 activator, aldo-keto reductase family member BIO activator, dihydrofolate reductase activator, pterin- 4-alpha-carbinolamine dehydratase activator, dihydropterine reductase activator, or combinations thereof.

BH4 biological activity agonists are readily available in the prior art. Preferably, the agonist is a tetrahydrobiopterin, such as sapropterin, 1 ', 2 ' -diacetyl-5, 6, 7, 8-tetrahydrobiopterin, or 6-methyl-5, 6, 7, 8-tetrahydrobiopterin, or any pterin analogue as described in US 3,557,106, US 7, 601,717, US 2010/0016328 A1 and US 8,324,210 (all incorporated herein by reference); or a trihydrobiopterin or a dihydrobiopterin; or a pharmaceutically acceptable salt thereof.

The BH4 biological activity agonist may also be any of BH4' s metabolic precursors, like a substrate of any one of the enzymes in the BH4 synthesis pathway mentioned above, like sepiapterin.

Furthermore, the BH4 biological activity agonist may be selected from the group consisting of 6-lactyl-7’ , 8’ -dihydropterin (sepiapterin), 6-1 ' , 2’ -dioxypropyl tetrahydropterin (6- pyruvoyltetrahydropterin) , 6-1’ -oxo-2’ -hydroxypropyl tetrahydropterin (6-lactoyltetrahydropterin) , and 6-1 ' -hydroxy-2’ - oxypropyl tetrahydropterin (6-hydroxypropy) tetrahydropterin) ,

(6R) -L-erythro-5, 6, 7, 8-tetrahydrobiopterin, (6R, S) -5, 6, 7, 8- tetrahydrobiopterin, 1’ , 2’ -diacetyl-5, 6, 7, 8-tetrahydrobiopterin, sepiapterin, 6-methyl-5, 6, 7, 8-tetrahydropterin, e-hydroxymethyls', 7, 8-tetrahydropterin, 6-phenyl-5, 6, 7, 8-tetrahydropterin and any other BH4 biological activity agonist disclosed in US

2006/0194808 Al, which is incorporated herein by reference; or a pharmaceutically acceptable salt thereof.

The BH4 biological activity agonist may also be one of the agonists disclosed in US 2003/0077335 Al (incorporated herein by reference), such as 7, 8-dihydroneopterin; 1’ -hydroxy-2’ - oxopropyltetrahydropterin; L-sepiapterin, 7, 8-dihydrobiopterin; pyruvoyltetrahydropterin; lactoyltetrahydropterin; or a pharmaceutically acceptable salt thereof.

Moreover, the BH4 biological activity agonist may be GCH1 activator. For instance, BH4 biological activity agonist may be a PPAR-a agonist such as fenofibrate (Liu, Jinbo, et al. "PPAR-a agonist fenofibrate upregulates tetrahydrobiopterin level through increasing the expression of Guanosine 5 ' -triphosphate cyclohydrolase-I in human umbilical vein endothelial cells."

PPAR research 2011 (2011).; incorporated herein by reference) or GW501516 (He, Tongrong, et al. "Activation of peroxisome prolif- erator-activated receptor-d enhances regenerative capacity of human endothelial progenitor cells by stimulating biosynthesis of tetrahydrobiopterin." Hypertension (2011); PMID 21709207; incorporated herein by reference) ; or a pharmaceutically acceptable salt thereof.

The BH4 biological activity agonist may also be any agonist disclosed in US 2010/00099997 Al (incorporated herein by reference) :

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 and Canadian application CA 2420374 (each incorporated herein by reference) each describe methods of making dihydrobiopterins, BH4 and derivatives thereof that may be used as BH4 biological activity agonist for the present invention.

In particular, U.S. Pat. No. 4,540,783 describes suitable BH4 biological activity agonists that are 1 ', 2 '-diacyl- (6R, S) - 5,6,7, 8-tetrahydro-L-biopterins, and inorganic or organic salts thereof, that are useful according to the therapeutic methods of the invention. Preferably pharmaceutically acceptable salts are used for therapeutic methods of the invention. The suitable BH4 biological activity agonists described in U.S. Pat. No.

4,540,783 have the general formula (I):

wherein R¹ and R² are the same or different and each is an acyl group . The acyl group has preferably 1 to 10 carbon atoms, in particular 3 to 10 carbon atoms. Preferably, the acyl group is represented by the general formula R⁵CO— wherein R⁵ is hydrogen or a hydrocarbon residue having 1 or more carbon atoms, in particular 2 to 9 carbon atoms . Preferable examples of the hydrocarbon residue represented by R⁵ are, for instance, a linear or branched alkyl group having 1 or more carbon atoms, preferably 2 to 9 carbon atoms, which is either saturated or unsaturated; a substituted or unsubstituted phenyl group represented by the general formula

wherein R⁶, R⁷, R⁸, R⁹ and R¹⁰ are hydrogen or a linear or branched alkyl group wherein the combined number of carbon atoms is R⁶,

R⁷, R⁸, R⁹, R¹⁰ is preferably not more than 3; a substituted or unsubstituted benzyl group represented by the general formula

wherein R¹¹ and R¹² are hydrogen, methyl or ethyl wherein the combined number of carbon atoms R¹¹ and R¹² is preferably not more than 2; and a substituted or unsubstituted arylalkyl group represented by the general formula

wherein R¹³ is hydrogen or methyl group. Among the above acyl groups, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl and benzoyl are most preferable. It is preferable that R¹ and R² are the same.

The compound of the general formula (I) has two diastere- omers, i.e. 1 ' , 2 '-diacyl- (6R) -5, 6, 7, 8-tetrahydro-L-biopterin and 1 ' , 2 '-diacyl- (6S) -5, 6, 7, 8-tetrahydro-L-biopterin which are dia- stereomeric at the 6 position. The compound for use as BH4 biological activity agonist of the invention can include either of the two diastereomers or a mixture thereof.

The compound (I) can be used as BH4 biological activity agonist in a form of an inorganic salt such as a hydrochloride, a sulfate or a phosphate, an organic salt such as an acetate, an oxalte, or a complex salt.

U.S. Pat. No. 4,550,109 describes suitable BH4 biological activity agonists that are lipoidal biopterins and tetrahydrobi- opterins. These lipoidal BH4 agonists may be administered as pharmaceutically acceptable salts according to the therapeutic and non-therapeutic methods of the invention. The compounds described in U.S. Pat. No. 4,550,109 are represented by the following structure:

wherein R is absent when there are two double bonds in ring B;

R is hydrogen when the two double bonds in ring B are absent; and R^r and R" are independently saturated or unsaturated, aliphatic hydrocarbon groups which are balanced in molecular weight such that they confer to the lipoidal compound a lipoidal property.

Generally, R^r and R" are selected from hydrocarbons having from 1 to 31 carbon units, with the limitation that the sum of carbon units of R^r+2R" is greater than 10 but less than 33.

In these BH4 biological activity agonists, the 2-N-acyl group is desirably from 9 to 32 and preferably 9 to 20 carbon units so as to confer lipoidal characteristics upon the final product. The 2-N-acyl group is exemplified by decanolyl-, pal- mitoyl-, stearoyl- and linoleyl. The 2-N-acyl group may be satu- rated as is stearoyl- or unsaturated as is linoleyl. In addition, non-toxic aromatic 2-N-acyl groups like phenylacetyl can also confer the desirable lipoidal characteristics to the final product. The 1 ' , 2 ' -di-O-acyl groups, are desirably lower molecular weight alkyls and alkenyls having from 2 to 8 and preferably 2 to 4 carbon units, with acetyl being exemplary.

U.S. Pat. No. 4,550,109 also describes suitable BH4 biological activity agonists of the formula:

O OA

wherein R is a naturally occurring fatty acid, which can be saturated or unsaturated, and Ac—COCH3. These agonists can be hydrogenated to form the corresponding tetrahydrobiopterins, which are useful according to the therapeutic methods of the invention. Exemplary chain lengths of the group R fatty acid range from CIO to C18 units. Exemplary agonists include 2N-Acetyl- 1 ' , 2 ' -di-O-Acetyl-L-Biopterin, 2-N-Decanoyl-l ' , 2 ' -di-O-acetyl-L- biopterin, 2-N-Palmitoyl-l ' , 2 ' -di-O-acetyl-L-biopterin, 2-N- Stearoyl-1 ' , 2 ' -di-O-acetyl-L-biopterin, 2-N-Linoleyl-l ' , 2 ' -di-O- acetyl-L-biopterin, and 2-N-Phenylacetyl-l ' , 2 ' -di-O-acetyl-L- biopterin, and the corresponding tetrahydrobiopterins .

Preferably the 1 ' , 2 '-diacyl- (6R, S) -5, 6, 7, 8-tetrahydro-L- biopterin or lipoidal tetrahydrobiopterin is selected as exhibiting at least 50% better oral bioavailability than BH4 ( ( 6R)— 5,6,7, 8-tetrahydro-L-biopterin) , for example, at least 15%, 20%, 25%, 30%, 35% or more oral bioavailability when taken on an empty stomach.

Furthermore, the BH4 biological activity agonist may be a mRNA coding for sepiapterin reductase, GTP cyclohydrolase 1, protein tyrosine phosphatase, aldo-keto reductase family member C3, aldo-keto reductase family member B10, dihydrofolate reductase, pter-in-4-alpha-carbinolamine dehydratase, and/or dihy- dropterine reductase. The mRNA may be a modified mRNA optimized and formulated for therapeutic delivery, as disclosed e.g. in US 2015/0017211 A1 which is incorporated herein by reference.

In certain embodiments, the BH4 biological activity agonist is a CRISPR/Cas system for introduction of mutations which increase expression or enzymatic activity of sepiapterin reductase, GTP cyclohydrolase 1, protein tyrosine phosphatase, aldo- keto reductase family member C3, aldo-keto reductase family member BIO, dihydrofolate reductase, pter-in-4-alpha-carbinolamine dehydratase, and/or dihydropterine reductase. Systems suitable therefor and methods related thereto are for instance disclosed in US 2016/0281111 Al, US 2017/0355985 A1 and US20170283830A1, all of which are incorporated herein by reference.

In preferred embodiments, the BH4 biological activity agonist is provided with microorganisms that recombinantly express the BH4 biological activity agonist. For this embodiment, the expressed agonist may be any one described above. Preferably in this embodiment, the agonist is tetrahydrobiopterin, such as sapropterin, 1 ', 2 ' -diacetyl-5, 6, 7, 8-tetrahydrobiopterin, or 6- methyl-5, 6, 7, 8-tetrahydrobiopterin, or any pterin analogue as described in US 3,557,106, US 7,601,717, US 2010/0016328 Al and US 8,324,210 (all incorporated herein by reference); or a trihy- drobiopterin or a dihydrobiopterin; or any of BH4' s metabolic precursors, like a substrate of any one of the enzymes in the BH4 synthesis pathway mentioned above, like sepiapterin. The microorganism is preferably pharmaceutical acceptable, such as a gut bacterium. It produces the agonist and provides a the patient or subject to be treated with it, e.g. by secretion into the gut from which the agonist is readily absorbed by the patient or subject to be treated. The microorganism may recombi- nantly express the 6-pyruvoyltetrahydropterin synthase (PTPS-2) . Recombinant expression means that an artificial copy of the agonist expression one or more gene (s) has been provided to the microorganism so that the gene's expression is enabled or increased as compared to the wild type microorganism of the same species and strain under the same experimental conditions, in particular in the gut . Example bacteria are disclosed in Belik, J. et al • / Intestinal microbiota as a tetrahydrobiopterin exoge- nous source in hph-1 mice. Sci . Rep. 7, (2017) . These include bacteria of the genus Adlercreutzia, Adlercreutzia, Bifidobacterium, Eggerthella, Gordonibacter, Microbacterium, Mobiluncus, Synechococcus; preferred are intestinal Actinobacteria bacteria and E. coli. Preferably the microorganism has the capacity to colonize the patient's gut, in particular the mammalian gut, even more preferred the human gut. These engineered microorganisms can then be used in any treatment of the invention, preferably in an anti-cancer therapy. The microorganism-derived BH4 agonist is more stable and is taken up better as compared to the chemical compound counterparts .

The BH4 biological activity agonist can be used with or provided in a pharmaceutical preparation. Preferably, the pharmaceutical preparation is in the form of a formulation for systemic treatment, e.g. parenteral (in particular intravenous) or oral. In embodiments, the BH4 biological activity agonist is the single active agent in the composition. It is also preferred that the composition does not comprise interleukin-2 (IL-2) .

A further embodiment is characterized in that the preparation is intended for oral intake, preferably in the form of pastilles, tablets, troches, lozenges, pills, gums, powders or drinking solutions . Systemic or topical distribution of a BH4 agonist 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, excipient, 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. Preferably, the preparation comprises buffers or pH adjusting

agents, e.g. selected from citric acid, acetic acid, fumaric acid, hydrochloric acid, malic acid, nitric acid, phosphoric acid, propionic acid, sulfuric acid, tartaric acid, or combinations thereof. A BH4 agonist can be in the form of a pharmaceutically acceptable salt, for example sodium salt, may also be used. Other pharmaceutically acceptable salts include, among others, potassium, lithium and ammonium salts. Preferred excipients are polymers, especially cellulose and cellulose derivatives.

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 especially preferred in case of large agonists, such as the mRNAs described above.

Preferably, a BH4 agonist is formulated for administration 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 preferred between 1 mg/kg and 40 mg/kg. The present invention also provides for the use of the pharmaceutical preparations.

The inventive BH4 biological agonist 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 agonist, 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. (Oligonucleotides. 2011 Feb; 21(1): 1-10) for this purpose, or an antibody, 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 antibody or nanobody, an antigen binding domain, etc... Zhou et al. described not only aptamers, but also means to bind therapeutic agents (here: BH4 biological activity agonists) 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 CDS . Zhou et al describe CD4-specific aptamers that are particularly preferred according to the invention. There reach high rates of internalization 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 comprising (i) a BH4 biological agonist and (ii) a T cell culturing medium and/or a T cell adsorbent and/or a T cell-specific drug delivery agent. These components (i) , any of (ii) are described above herein.

The present invention is further illustrated by the following 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) depicting early activation markers (CD25, CD62L) and IL-2 secretion (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 after 3 days from control and Gchl;Lck mice. Representative experiment 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 proliferation 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 GcM-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 infiltration of various immune cells (CD4⁺ and CD3⁺ T cells, CDllc⁺ dendritic cells and MPO⁺ neutrophils) . Scale bar, 200pm. c, Allergic airway inflammatory disease model and quantification of inflammatory cells in bronchoalveolar lavage fluids (BALFs) . Data 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, Percentage increase of ear swelling after re-challenge using the 2,4, 6-trinitrochlorobenzene (TNCB) -dependent skin hypersensitivity 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 repeated >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 multiple 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 individual 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 representative 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 inflammatory 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 measurements 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, Representative oxygen consumption traces from permeabilized cells from 10 hour activated CD4⁺ T cells from (g) control and Gchl;Lck mice and (h) activated wild type mice treated with vehicle or SPRi3 (50mM) . i, Relative Complex I and II activities (mean values ± 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 control, GCH1 ;RORc mice as well as control cells treated with SPRi3 (50mM) . Experiments were repeated 3 times showing comparable results. 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 histograms are shown on the left and quantification (mean ± s.e.m.) on the right. ****P < 0.0001 (One-way ANOVA with Tukey' s multiple 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 depicting 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, Representative 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 repeated 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/200pL/mouse) tumor cells were injected into the mammary fat pad of syngeneic C57B16 mice and tumor sizes monitored over time, b, Effect of BH4 supplementation 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 administered. 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 multiple comparisons) . c, Quantification of intratumoral effector T cells (CD44⁺CD62L¹⁰) assayed from E0071 tumors on day 28 of vehicle 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 activated CD4⁺ T cells treated with kynurenine (50mM) and BH4 (IOmM) . 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 vehicle (DMSO) , kynurenine (KYN) alone (50mM) or KYN (50mM) plus BH4 (IOmM) for 10 hours. Experiments were repeated 3 times showing 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 depicting 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 various 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 (Student's t-test). i, Death by neglect of purified CD4⁺ T cells cultured without stimulation for up to 56 hours. Data are shown as means ± s.e.m. n=3 for each genotype. NS, not significant (Student'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;MBl mice depicting B cell developmental populations . b, c, Representative FACS histogram depicting LPS (lpg/ml) -stimulated B cell proliferation from control and Gchl;MBl 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;MBl mice stimulated with LPS (20pg/ml) for 5 days inducing class switch recombination to IgG3. FACS blots are representative of two independent experiments .

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 (lOOpg 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, Representative FACS blots depicting activation marker profiles of purified 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 serving as antigen presenting cells (APCs) plus soluble anti-CD3 antibody (lpg/ml) and treated with vehicle (DMSO), SPRi3 (50mM) or SP (5mM) . FACS blots are representative of three independent experiments, 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 genotype/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 (4pg/ml+2pg/ml) stimulated for 12, 24 and 72 hours, b, representative histogram showing iNOS expression in control and GcM-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) . Peritoneal, 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 (TCR3⁺) 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). b, Proportion of CD4⁺ and CD8⁺ T cells among the splenic T cell (TCR3⁺) 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 significant. (Student's t-test). e, Proportion of CD4⁺ and CD8⁺ naive (CD44¹⁰; 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 overexpression in vitro. b,c, Quantification of 4-OHT-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 reductase. 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 stimulation. ***P < 0.001 (One-way ANOVA with Dunnett's multiple comparisons test) . g, Representative histograms depicting proliferation of wild type control CD4⁺ T cells after 3 days of anti- CD3/CD28 stimulation which were vehicle (DMSO) treated or supplemented with BH4 (10mM) from control and Gchl ;RORc mice. FACS blots are representative of two independent experiments.

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 GCH1-HA overexpression cassette to induce BH4 overproduction and Gchl floxed mice prevented BH4 production have been previously reported (Chuaiphichai, S. et al. Hypertension 64, 530-540 (2014)). For both gain- and loss-of-function experiments, we bred GCH1-HA and Gchl floxed mice to the T cell- specific lines LCK-Cre, CD4-Cre, RORgammact-Cre or the ubiquitous 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 concentration of 10mM and SPRi3 at a concentration of 50mM unless otherwise stated in the figure legends. For in vivo use, BH4 was reconstituted in sterile saline under argon gas. Kynurenine (# K8625) and NAG (# A9165) were purchased from Sigma.

Determination of BH4 levels. BH4 (tetrahydrobiopterin) , and oxidized biopterins (BH2 and biopterin, ) were determined by high- performance liquid chromatography (HPLC) followed by electrochemical 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^eC, supernatant was removed and ice- cold acid precipitation buffer (1 M phosphoric acid, 2 M trichloroacetic acid, 1 mM dithioerythritol) added. Following centrifugation at 13,200 rpm for 10 min at 4^eC, 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 10^s 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 collected 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 cultured with purified splenic dendritic cells and soluble anti-CD3 antibody (lpg/ml) for three days. Similarly, B cells were purified using microbeads (CD19⁺; Miltenyi Biotec) from the spleen, loaded with cell tracer, stimulated with LPS (lpg/ml) and analyzed for proliferation as described above. For class switch recombination 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 using the Click-iT® EdU Flow Cytometry Cell Proliferation Assay (Invitrogen) . Briefly, purified CD4⁺ T cells were activated with anti-CD3 (4pg/ml) and anti-CD28 (2pg/ml) as described above. EdU was pulsed into the wells for 4 hours after 16hrs of stimulation. The cells were prepared and stained with EdU as per the manufacturer's instructions.

Mitochondrial respiration and metabolomics . Mitochondrial respiratory parameters were measured with high-resolution respirometry (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^eC (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 induced 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 Seahorse technology. To measure ATP, purified T cells were either left unstimulated or stimulated with plate-bound anti-CD3

(4pg/ml) and anti-CD28 (2pg/ml) for the times indicted in the figure. ATP was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) . To determine ROS levels, purified T cells were activated with anti-CD3 plate-bound anti-CD3 (4pg/ml) and anti-CD28 (2pg/ml) for 10 hours. Cells were washed once with HESS and stained in IOmM DHE (Invitrogen) for 30 mins at 37°C. Cells were washed 2X with HESS and assayed by flow cytometry. Profiling of biogenic amines by hydrophilic interaction liquid chromatography (HILIC-QTOF) mass spectrometry was performed 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 reagent 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 orthotopically injected into the fat pad of the fourth mammary gland (2.5x10^s cells/200pL/mouse) . BH4 administration was delivered i.p. (lOOmg/kg) after tumors were palpable (day 10) and treatment was continued for 7 days. Tumors were measured using digital 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.

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

Protein blotting. Protein blotting was carried out using standard protocols. Blots were blocked for 1 hour with 5% BSA in TEST (lx TBS and 0.1% Tween-20) and were then incubated overnight at 4^eC with primary antibodies (See Table 1), diluted in 5% BSA in TEST (1:1,000 dilution). Blots were washed three times in TEST for 15 min and were then incubated with HRP-conjugated secondary antibodies (1:2,500 dilution; GE Healthcare, NA9340V) for 45 min at room temperature, washed three times in TEST 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 Zhfiiga-Pflucker; University of Toronto) were maintained as described 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 TOHb^~TOHgd^~OΏ4^~OΏ8a^~CD28CD25^hiCD44 -/lo 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 medium 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 aMEM 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 . 5x10^s MACS-purified naive CD4⁺CD62L⁺ T cells from control and GCHl;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 colitis 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 fluorescence 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 sequential incubation with methanol, avidin/biotin (Vector Laboratories) , and protein blocking reagent (DAKO) to eliminate unspecific 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 manufacturer's instructions (Perkin Elmer). Before examination, nuclei were counterstained with Hoechst 3342 (Invitrogen) .

OVA immunization and Airway hyperresponsiveness. For OVA immunization study, immunization was performed using 100pg OVA per mouse in 200pL Alum intraperitoneally (i.p.). Blood was collected 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 rechallenge responses. For measurements of lung function, deeply anesthetized mice (pentobarbital (60 mg/kg) underwent a tracheotomy 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 pressure was set at 2 cm H₂0. Lung resistance and elastance of the respiratory system was determined in response to in-line aerosolized 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 supernatant was discarded and cells resuspended in 200 mΐ. Bronchoalveolar 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 lymphocytes as F4/80^Neg-Ly6g^Neg-CD3^Pos . Total BAL cell counts were performed using a standard hemocytometer, with absolute cell numbers calculated as total BAL cell number multiplied by the percentage 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 applying 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 MOG35-55 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 expression 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 assayed as previously described (Arai, N • / et al. Pediatrics 70, 426-30 (1982)). The enzymatic conversion of qBH2 to BH4 was followed 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 intervals in 200m1 buffer containing 50mM FICC, ImM 6- methyltetrahydropterin (6MPH4), 20nM DHPR, 50mM NADH and selected inhibitors. A control lacking DHPR was ran in parallel to assess 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, H₂0) [L -mol^-1 -cm^-1 ] . Generally, 50mM of FOCC and FICC in buffer were measured in isolated 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, intracellular iron measurements were carried out by using a Perki- nElmer Analyst 800 equipped with a transversely heated graphite atomizer (THGA) . A Zeeman-effeet background correction was realized by a 0.8 T magnetic field, oriented longitudinally with respect 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 initial 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 (NH₄) ₂Fe (S0₄) 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 analyses. 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 atomizer in randomized fashion as triplicates, using a PerkinElmer AS-800 autosampler with an injection volume of 20 mΐ. The solvent was evaporated by a slow temperature gradient to 130 ^eC, ashing took place at a maximum temperature of 1,000 ^eC, and the atomization profile was read at 2,000 ^eC. 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 ^eC. The integrity of each analysis was verified by a visual inspection of the respective time-dependent atomization profile.

Human T cell proliferation assays. Proliferation of peripheral blood mononuclear cells (PBMCs) , obtained from healthy blood donors, 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 thymidine was measured by liquid scintillation spectroscopy. In addition, we also determined the proliferation of alloreactive human T cells. PBMCs from a healthy donor were stimulated with M21 tumor cells. Alloreactive T cells-based on MHC mismatch were cultured 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 ¹ s post-hoc test for multiple comparisons was used for analysis between multiple 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 isolated CD4⁺ and CD8⁺ T cells from our Gchl-Gfp reporter mouse line

(Latremoliere, A. et al • / Neuron 86, 1393-1406 (2015)), to report 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 expression 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 crossing 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 GCH1-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 populations 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 stimulation, we evaluated antigen receptor signaling in mature peripheral T cell activation. Following TCR engagement, we observed no differences between Gchl-null T cells and control cells in either surface activation marker expression or in interleukin (IL) -2 secretion after 16 hours of TCR (anti-CD3/CD28) stimulation (Fig. If), suggesting that early events in T cell activation 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 markedly reduced capacity to proliferate (Fig. lg, h) . In contrast to peripheral T cells, Gchl ablation did not affect proliferation of DN3a thymocytes in response to signals from co-cultured 0P9-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, peripheral 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 proliferation (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 development 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 unchecked donor T cells activate and proliferate in response to the intestinal microflora, orchestrating an intestinal inflammatory 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 intestinal damage and colitis (Fig. 2a) . Moreover, transfer of Gchl- ablated CD4⁺ T cells triggered a dramatically lower influx of immune 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 13,14 . Allergic inflammation 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) . Compared 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 challenge (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 encephalomyelitis (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 neurological 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 alleviates 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 pharmacologically 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 terminal 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 associated 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 stimulation (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 control 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 significantly ameliorated colitis, and greatly diminished the intestinal 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 proliferative 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 proliferation in mouse and human and, importantly, can be pharmacologically 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 acid hydroxylases which are required for the synthesis of serotonin, 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 corresponding amino acid precursors, in resting and activated T cells, and in the supernatant after TCR-stimulation. T cells from control and GcM-mutated animals either showed no expression 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 expression 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 accession number GSE108101) . Intriguingly, detailed analysis uncovered 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 GcM-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 particular for complex I and complex II of oxidative phosphorylation 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 molecular 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) . Importantly, total iron levels were significantly reduced in TCR- activated GcM-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 facilitates the reduction of ferric-cytochrome C to its ferrous form, suggested that BH4 deficiency may result in mitochondrial dysfunction. 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 significantly lower in both GcM-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 oxygen species (ROS) levels were significantly elevated in activated CD4⁺ T cells from GcM-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 GcM-ablated T cells (Fig. 4k), suggesting 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 levels, inducible NOS (iNOS) becomes uncoupled and generates superoxide at the expense of NO and thus may be the source of the elevated 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 mechanisms, our data show that antigen receptor stimulated, BH4- depleted T cells display defective iron metabolism and mitochondrial dysfunction.

Example 6: Enhanced BH4 production super-activates T cells

To investigate whether elevated GCH1 enhances T cell function in vivo, we crossed the Lck-Cre driver line to Cre- recombinase dependent, GCH1 over-expressing mice (hereafter referred 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 activated 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 confirmed 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 effect of depleting GCH1/BH4, leading to enhanced T cell proliferation and function.

We next asked whether pharmacological elevation of BH4 enhanced T cell function. Sepiapterin (SP) is a metabolite of the BH4 salvage pathway distinct from the GCH1-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) . Furthermore, treatment of stimulated CD4⁺ T control cells with BH4 itself dramatically increased both proliferation and IL-2 secretion (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 • f et al. Nat. Rev. Cancer 11, 805-12 (2011)). Additional 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 tumors, which were then monitored over several weeks. GOE_/CD4 mice, unlike controls, completely rejected tumor growth (Fig.

6a) . Moreover, therapeutic treatment of mice carrying established 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 administration increased T cell activation and enhanced their antitumor response.

Given the anti-tumor activity of GCH1/BH4 in T cells we hypothesized 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 energy.

It has been proposed that IDO-produced kynurenine metabolites directly induce immunosuppression via increased transdifferentiation of CD4⁺ T cells into Tregs, as well as via activation of the aryl hydrocarbon receptor (AhR) on dendritic cells and macrophages 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 acid 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, importantly, the proliferative capacity was fully restored by addition 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 inhibition of the BH4 synthetic pathway. Thus, BH4 enhances antitumor activity of T cells and counteracts kynurenine-dependent immunosuppressive effects.

Discussion

T cells play an essential role in combatting invading pathogens as well as providing anti-cancer immunity. Conversely, self-reactive T cells can cause devastation manifesting in autoimmune diseases. Emerging data highlight the intimate relationship between T cell function and cellular metabolism. Identifying pathways that coordinate metabolic processes with inflammatory effector functions is of paramount therapeutic importance to not only enhance T cell function in the case of cancer immunotherapy, but, equally crucial, to repress their function under conditions of autoimmunity. Here, we have identified that the BH4 pathways, GCH1 and its 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 mitochondrial 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 allergies in several different T cell-mediated model systems. Conversely, T cell specific overexpression of Gchl and administration of BH4 markedly enhanced anti-cancer immunity in an orthotopic breast cancer model, supporting the notion that the BH4 pathway is an integral checkpoint control in T cell proliferation. The tryptophan metabolite kynurenine, generated by the enzyme 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. Indeed, 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 effective 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 GcM-ablated T cells have lower iron content after activation. BH4 can directly reduce ferric iron to ferrous iron, including reduction of cytochome-c-Fe³⁺ to cytochrome-c-Fe²⁺, affecting 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 proliferation. 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. Importantly, 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. Moreover, several studies in animals and humans have shown that nutritional 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 proliferation.

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 appears to be a link between the local immunosuppressive tumor environment 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, blockade 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 increasing T cell activity comprising increasing 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 cell is a recombinant T cell, preferably a chimeric antigen receptor (CAR) T cell or a cell with a recombinant T cell receptor (TCR) .

4. The method of any one of claims 1 to 3, wherein said T cells are from a patient having or predisposed of developing a cancer.

5. The method of any one of claims 1 to 4, wherein said cancer is selected from breast cancer, lung cancer, ovarian cancer, cervical cancer, hepatocellular carcinoma, renal cell carcinoma, thyroid cancer, neuroendocrine cancer, gastro-oesophageal cancer, bladder cancer, colorectal cancer, glioma, prostate cancer, pancreatic cancer, stomach cancer, oesophagal cancer, liver cancer, melanoma, glioblastoma, leukemia and lymphoma.

6. The method of any one of claims 1 to 5, comprising treating the T cell with a BH4 biological activity agonist.

7. The method of any one of claims 4 to 6, comprising diagnosing the patient with the cancer or a predisposition thereto and then administering to the patient a BH4 biological activity agonist .

8 . The method of any one of claims 1 to 7, wherein said T cells are sensitized against a tumour antigen, preferably wherein the method comprises detecting said sensitized T cells in a patient and/or obtaining said sensitized T cells from the patient.

9. The method of any one of claims 6 to 8, wherein the BH4 biological activity agonist is selected from a sepiapterin reductase activator, GTP cyclohydrolase 1 activator, protein tyrosine phosphatase activator, aldo-keto reductase family member C3 ac- tivator, aldo-keto reductase family member BIO activator, dihydrofolate reductase activator, pterin-4-alpha-carbinolamine dehydratase activator, dihydropterine reductase activator, or combinations thereof.

10. The method of any one of claims 5 to 9, wherein the BH4 biological activity agonist is selected from:

a tetrahydrobiopterin, such as sapropterin, 1¹ , 2¹ -diacetyl-

5, 6, 7, 8-tetrahydrobiopterin, and 6-methyl-5, 6, 7, 8- tetrahydrobiopterin, and a trihydrobiopterin and a dihydrobiop- terin, and sepiapterin, 6-pyruvoyltetrahydropterin, 6- lactoyltetrahydropterin, 6-hydroxypropy) tetrahydropterin, (6R)-

L-erythro-5, 6, 7, 8-tetrahydrobiopterin, (6R, S) -5, 6, 7, 8- tetrahydrobiopterin, 1 ' , 2 ' -diacetyl-5, 6, 7, 8-tetrahydrobiopterin,

6-methyl-5, 6, 7, 8-tetrahydropterin, 6-hydroxymethyl-5, 6,7,8- tetrahydropterin, 6-phenyl-5, 6, 7, 8-tetrahydropterin, 7,8- dihydroneopterin, 1 ' -hydroxy-2 ' -oxopropyltetrahydropterin, L- sepiapterin, 7, 8-dihydrobiopterin, pyruvoyltetrahydropterin, lactoyltetrahydropterin, and a compound of the general formula

wherein R¹ and R² are the same or different and each is an acyl group, and

a compound of the general formula

wherein R is absent when there are two double bonds in ring B, R is hydrogen when the two double bonds in ring B are absent, and R^r and R” are independently saturated or unsaturated, ali- phatic hydrocarbon groups and

a compound of the general formula

wherein R is a naturally occurring fatty acid, which can be saturated or unsaturated, and Ac—COCH₃;

or pharmaceutically acceptable salts thereof; and

a PPAR-a agonist such as fenofibrate or GW501516; or pharmaceutically acceptable salts thereof; and

an mRNA coding for sepiapterin reductase, GTP cyclohydrolase 1, protein tyrosine phosphatase, aldo-keto reductase family member C3, aldo-keto reductase family member BIO, dihydrofolate reductase, pter-in-4-alpha-carbinolamine dehydratase, and/or dihy- dropterine reductase; and

a CRISPR/Cas system for introduction of mutations which increase expression or enzymatic activity of sepiapterin reductase, GTP cyclohydrolase 1, protein tyrosine phosphatase, aldo-keto reductase family member C3, aldo-keto reductase family member BIO, dihydrofolate reductase, pter-in-4-alpha-carbinolamine dehydratase, and/or dihydropterine reductase.

11 . The method of any one of claims 1 to 10, wherein the patient does not have and/or is not predisposed to a condition selected from neurological conditions, psychiatric conditions, such as depression, and metabolic conditions such as phenylketonuria and tetrahydrobiopterin deficiency.

12. The method of any one of claims 1 to 11, wherein increasing BH4 biological activity in said T cell is in vitro and/or ex vivo, preferably of isolated and/or purified T cells; preferably further comprising introducing or reintroducing the treated T cell into the patient.

13. A BH4 biological activity agonist for use in the prevention or treatment of a cancer, preferably wherein said prevention or treatment comprises a method of any one of claims 1 to 12 and/or preferably wherein said prevention or treatment is in combination with a therapy with an immune checkpoint inhibitor.

14. Sapropterin, sepiapterin, or a pharmaceutically acceptable salt thereof for use in the treatment of a cancer in a patient, 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 activity agonist and (ii) a T cell culturing medium and/or a T cell adsorbent and/or a T cell-specific drug delivery agent.