Inhibition of Acetyl-CoA Metabolism for Treatment and Prevention of Immune System Diseases and Disorders
1. A method of treating or preventing a T cell-mediated disease or disorder, the method comprising administering to a subject having or susceptible to developing a T cell-mediated disease or disorder a composition comprising an effective amount of a lactate dehydrogenase A (LDHA) inhibitor.
Provided are methods for treating or preventing T cell-mediated diseases or disorders by administering an inhibitor of acetyl-CoA production, such as an inhibitor of lactate dehydrogenase A or an inhibitor of ATP-citrate lyase.
- 1. A method of treating or preventing a T cell-mediated disease or disorder, the method comprising administering to a subject having or susceptible to developing a T cell-mediated disease or disorder a composition comprising an effective amount of a lactate dehydrogenase A (LDHA) inhibitor.
- 4. A method of treating or preventing a T cell-mediated disease or disorder, the method comprising administering to a subject having or susceptible to developing a T cell-mediated disease or disorder a composition comprising an effective amount of an ATP-citrate lyase (ACL) inhibitor.
- 13-18. -18. (canceled)
This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/394,859, filed on Sep. 15, 2016, the entire contents of which are incorporated by reference.
This invention was made with government support under grant number CA008748, awarded by the National Institutes of Health. The government has certain rights in the invention.
A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
For countries that permit incorporation by reference, all of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers'"'"' instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.
T cell activation and differentiation are associated with metabolic rewiring (1-4). A metabolic hallmark of activated T cells is aerobic glycolysis (the Warburg effect) (5), the conversion of glucose to lactate in the presence of oxygen. However, its physiopathological functions remain incompletely understood (6-8). As the major carbon source, glucose plays important roles in T cell development, proliferation, and function (9-15). Aerobic glycolysis has been implicated in augmenting effector T cell responses, including expression of the pro-inflammatory cytokine interferon (IFN)-γ, via 3′ untranslated region (3′UTR)-mediated mechanisms. However, the specific contribution of aerobic glycolysis to T cell responses has not been well defined. Using galactose as a sugar source, aerobic glycolysis was proposed to support IFN-γ expression through 3′UTR-mediated mechanisms (12). Although galactose is metabolized at a slower rate than glucose via the Leloir pathway, both sugars are converted to lactate (16), rendering the galactose system unable to model aerobic glycolysis deficiency in a definitive manner.
By converting pyruvate to lactate with regeneration of nicotinamide adenine dinucleotide (NAD+) (17), lactate dehydrogenase (LDH) defines the biochemical reaction of aerobic glycolysis. LDHA and LDHB form five tetrameric LDH isoenzymes (A4B0, A3B1, A2B2, A1B3, and A0B4) with distinct kinetic properties (17). ATP-citrate lyase (ACL) is an enzyme converting citrate to acetyl-CoA in the cytosol.
A better understanding of LDH and ACL activity upon T cell activation could have therapeutic implications for diseases and disorders of the immune system, including autoimmunity and transplant rejection.
Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.
The inventors show herein that LDHA is induced in activated T cells to support aerobic glycolysis, but promotes IFN-γ expression independently of its 3′UTR. Instead, LDHA maintains high levels of acetyl-CoA to enhance histone acetylation and transcription of Ifng. Ablation of LDHA in T cells protects mice from immunopathology triggered by excessive IFN-γ expression or deficiency of regulatory T cells. These findings reveal an epigenetic mechanism by which aerobic glycolysis promotes effector T cell differentiation, and indicate that LDHA and downstream enzymes such as ACL can be targeted therapeutically in diseases or disorders associated with T cell activation and/or differentiation and/or proliferation, such as autoinflammatory and autoimmune diseases, graft rejection, and transplant rejection.
Accordingly, the invention provides a method of treating or preventing a T cell-mediated disease or disorder, the method comprising reducing acetyl-CoA production in a subject having or susceptible to developing a T cell-mediated disease or disorder.
In one aspect, the invention provides a method of treating or preventing a T cell-mediated disease or disorder, the method comprising administering to a subject having or susceptible to developing a T cell-mediated disease or disorder a composition comprising an effective amount of a lactate dehydrogenase A (LDHA) inhibitor. In another aspect, the invention provides a composition comprising an LDHA inhibitor for use in the treatment of a T cell-mediated disease or disorder. The invention also provides a composition comprising an LDHA inhibitor for the manufacture of a medicament for use in the treatment of a T cell-mediated disease or disorder. In some embodiments, the LDHA inhibitor is selected from the group consisting of FX11, Galloflavin, GNE-140, GSK 2837808A, and NHI 2. In a preferred embodiment, the LDHA inhibitor is FX11.
In a further aspect, the invention provides a method of treating or preventing a T cell-mediated disease or disorder, the method comprising administering to a subject having or susceptible to developing a T cell-mediated disease or disorder a composition comprising an effective amount of an ATP-citrate lyase (ACL) inhibitor. In yet another aspect, the invention provides a composition comprising an ACL inhibitor for use in the treatment of a T cell-mediated disease or disorder. The invention also provides a composition comprising an ACL inhibitor for the manufacture of a medicament for use in the treatment of a T cell-mediated disease or disorder. In some embodiments, the ACL inhibitor is selected from the group consisting of BMS 303141, ETC-1002, and SB 204990. In a preferred embodiment, the ACL inhibitor is BMS 303141.
In some embodiments, the composition is administered orally. In some embodiments, the composition is administered intravenously.
In a preferred embodiment, the subject is a human.
T cell-mediated diseases or disorders that can be treated or prevented by the methods of the invention include, but are not limited to, autoimmune disorders, graft rejection, inflammation, and organ rejection. In certain embodiments, the autoimmune disorder is selected from the group consisting of graft-versus-host disease (GVHD), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis, rheumatoid arthritis, systemic lupus erythematosus (SLE), and type-1 diabetes. In one embodiment, the composition is administered prophylactically within about 24 hours of an organ transplant or tissue graft.
We demonstrate that genes involved in aerobic glycolysis, such as lactate dehydrogenase A (LDHA), also play a role in T cell differentiation by an epigenetic mechanism, which is distinct from how they were thought to act. Deficiency of this enzyme (or inhibition of the enzyme) reverses the symptoms of immune disorders seen in the Scurfy mouse model. Scurfy mice have defective T cell tolerance leading to an X-linked lymphoproliferative disease that parallels the X-linked autoimmunity-allergic disregulation syndrome (XLAAD) in humans. This work has identified a critical role for LDHA-mediated aerobic glycolysis in promoting autoreactive Th1 and Th17 cell responses. LDHA inhibitors are being developed to target tumor cell metabolism. Our data indicate that LDHA inhibitors are immunosuppressive, and thus may complicate their applications in cancer. Instead, LDHA inhibitors, as well as inhibitors targeting other enzymes involved in acetyl-CoA metabolism, including ACL, can be useful in treatment of diseases and disorders associated with T cell activation, differentiation, or expansion.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. For example, The Dictionary of Cell and Molecular Biology (5th ed. J. M. Lackie ed., 2013), the Oxford Dictionary of Biochemistry and Molecular Biology (2d ed. R. Cammack et al. eds., 2008), and The Concise Dictionary of Biomedicine and Molecular Biology (2d ed. P-S. Juo, 2002) can provide one of skill with general definitions of some terms used herein.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.
Furthermore, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).
Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10. Where a numeric term is preceded by “about,” the term includes the stated number and values ±10% of the stated number. The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
Amino acids are referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation, and nucleic acid sequences are written left to right in 5′ to 3′ orientation.
Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are included.
The term “T cell-mediated disease or disorder,” as used herein, includes diseases and disorders characterized by T cell activation, T cell differentiation, and/or T cell proliferation. T cell-mediated inflammation, graft or organ rejection, and autoimmune diseases, such as graft-versus-host disease (GVHD), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis, rheumatoid arthritis, systemic lupus erythematosus (SLE), and type-1 diabetes, are examples of T cell-mediated diseases or disorders.
An “active agent” is an agent which itself has biological activity, or which is a precursor or prodrug that is converted in the body to an agent having biological activity. Active agents useful in the methods of the invention include inhibitors of acetyl-CoA production, such as LDHA inhibitors and ACL inhibitors.
An “LDHA inhibitor” is an active agent that agonizes or antagonizes the activity of lactate dehydrogenase A or reduces its production in a cell. Examples of LDHA inhibitors that are suitable for use in the present invention include FX11, Galloflavin, GNE-140, quinoline 3-sulfonamides (38), including GSK 2837808A, and NHI 2. “FX11” refers to a compound having the structure:
“Galloflavin” refers to 3,8,9,10-tetrahydroxy-pyrano[3,2-c]benzopyran-2,6-dione. “GNE-140” refers to (2R)-5-(2-chlorophenyl)sulfanyl-4-hydroxy-2-(4-morpholin-4-ylphenyl)-2-thiophen-3-yl-1,3-dihydropyridin-6-one. “GSK 2837808A” refers to 3-[[3-(cyclopropylsulfamoyl)-7-(2,4-dimethoxypyrimidin-5-yl)quinolin-4-yl]amino]-5-(3,5-difluorophenoxy)benzoic acid. “NHI 2” refers to methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate. Derivatives of these compounds that act as LDHA inhibitors, and their pharmaceutically acceptable salts, are also suitable for use in the invention.
An “ACL inhibitor” is an active agent that agonizes or antagonizes the activity of ATP-citrate lyase or reduces its production in a cell. Examples of ACL inhibitors that are suitable for use in the present invention include BMS 303141, ETC-1002, MEDICA 16, and SB 204990. “BMS 303141” refers to 3,5-dichloro-2-hydroxy-N-(4-methoxy[1,1′-biphenyl]-3-yl)-benzenesulfonamide. “ETC-1002” refers to 8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid. “MEDICA 16” refers to 3,3,14,14-tetramethylhexadecanedioic acid. “SB 204990” refers to (3R,5S)-rel-5-[6-(2,4-dichlorophenyl)hexyl]tetrahydro-3-hydroxy-2-oxo-3-furanacetic acid.
The terms “inhibit,” “block,” and “suppress” are used interchangeably and refer to any statistically significant decrease in biological activity, including full blocking of the activity.
By “subject” or “individual” or “patient” is meant any subject, preferably a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and so on.
Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder. In certain embodiments, a subject is successfully “treated” for a disease or disorder according to the methods provided herein if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder.
“Prevent” or “prevention” refers to prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of prevention include those at risk of or susceptible to developing the disorder. Subjects that are at risk of or susceptible to developing a T cell-mediated disease or disorder include, but are not limited to, patients receiving a tissue graft or an organ transplant, patients having a genetic predisposition to a T cell-mediated disease or disorder, and patients having another disease or disorder of the immune system, including another T cell-mediated disease or disorder. In certain embodiments, a disease or disorder is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder, than a patient who has not been subject to the methods of the invention.
In a prophylactic context, a composition for inhibiting acetyl-CoA production, such as a composition comprising an LDHA inhibitor or an ACL inhibitor, can be administered at any time before or after an event, for example, a tissue graft or an organ transplant, which places a subject at risk of or susceptible to developing a T cell-mediated disease or disorder. In some aspects, the composition for inhibiting acetyl-CoA production is administered prophylactically up to about one week before the event, such as 1, 2, 3, 4, 5, 6, or 7 days before the event. In some instances, the composition for inhibiting acetyl-CoA production is administered prophylactically on the same day as the event. In some embodiments, the pharmaceutical composition is administered prophylactically within 7 days of the event, for example, within about 1, 2, 3, 4, 5, 6, or 7 days. In a preferred embodiment, the composition for inhibiting acetyl-CoA production is administered within 24 hours of the event, i.e., about 24 hours before the beginning of the event to about 24 hours after the completion of the event.
The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Pharmaceutical compositions can be in numerous dosage forms, for example, tablet, capsule, liquid, solution, softgel, suspension, emulsion, syrup, elixir, tincture, film, powder, hydrogel, ointment, paste, cream, lotion, gel, mousse, foam, lacquer, spray, aerosol, inhaler, nebulizer, ophthalmic drops, patch, suppository, and/or enema. Pharmaceutical compositions typically comprise a pharmaceutically acceptable carrier, and can comprise one or more of a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), a stabilizing agent (e.g. human albumin), a preservative (e.g. benzyl alcohol), a penetration enhancer, an absorption promoter to enhance bioavailability and/or other conventional solubilizing or dispersing agents. Choice of dosage form and excipients depends upon the active agent to be delivered and the disease or disorder to be treated or prevented, and is routine to one of ordinary skill in the art.
“Systemic administration” means that a pharmaceutical composition is administered such that the active agent enters the circulatory system, for example, via enteral, parenteral, inhalational, or transdermal routes. Enteral routes of administration involve the gastrointestinal tract and include, without limitation, oral, sublingual, buccal, and rectal delivery. Parenteral routes of administration involve routes other than the gastrointestinal tract and include, without limitation, intravenous, intramuscular, intraperitoneal, intrathecal, and subcutaneous. “Local administration” means that a pharmaceutical composition is administered directly to where its action is desired (e.g., at or near the site of the injury or symptoms). Local routes of administration include, without limitation, topical, inhalational, subcutaneous, ophthalmic, and otic. It is within the purview of one of ordinary skill in the art to formulate pharmaceutical compositions that are suitable for their intended route of administration.
An “effective amount” of a composition as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose, route of administration, and dosage form.
In some embodiments, administration of the LDHA inhibitor or the ACL inhibitor can comprise systemic administration, at any suitable dose and/or according to any suitable dosing regimen, as determined by one of skill in the art. The LDHA inhibitor or the ACL inhibitor can be administered according to any suitable dosing regimen, for example, where the daily dose is divided into two or more separate doses. It is within the skill of the ordinary artisan to determine a dosing schedule and duration for administration. In some embodiments, the pharmaceutical composition is administered orally at least once a day or at least twice a day. In some embodiments, the pharmaceutical composition is administered intravenously at least once a day or at least twice a day. In some embodiments, the pharmaceutical composition is administered subcutaneously at least once a day or at least twice a day.
Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.
To characterize LDH activity in activated CD4+ cells, we performed a zymography assay. Activated CD4+ T cells manifested LDH activity predominantly in the form of A4B0, similar to that of muscle tissues (
To study the definitive function of aerobic glycolysis, we deleted LDHA specifically in T cells (CD4CreLdhafl/fl, designated as knock-out, KO) (
13C-isotope labeled glucose (13C6-glucose) tracing experiments showed that glycolysis was slowed down at the GAPDH step in KO T cells (
LDHA deficiency did not affect thymic development of conventional or regulatory T (Treg) cells (
Glycolysis promotes expression of effector molecules including the type 1 cytokine IFN-γ (9, 12-15). Indeed, LDHA deficiency led to diminished IFN-γ expression in T cells differentiated under T helper 1 (Th1) conditions (
To explore the definitive function of 3′UTR in LDHA control of IFN-γ expression, we used an Ifng reporter allele Yeti (yellow-enhanced transcript for IFN-γ) (20), in which the Ifng 3′UTR was replaced by 3′UTR of the bovine growth hormone (BGH) gene (
To determine whether reduced IFN-γ production in KO T cells was caused by diminished transcription, we performed RNA sequencing experiments. 363 transcripts were differentially expressed between WT and KO Th1 cells, among which 220 transcripts, including Ifng, were downregulated in KO T cells (
Glucose metabolism is implicated in the control of gene expression through epigenetic mechanisms including histone acetylation (7, 22). We performed chromatin immunoprecipitation-sequencing (ChIP-seq) analysis of histone H3 acetylation at the lysine 9 residue (H3K9Ac), a histone mark associated with active transcription. The ChIP-seq analysis showed that the differentially expressed genes between WT and KO Th1 cells had varying levels of H3K9Ac (
Compared to the constitutively active Cd3e locus, diminished H3K9Ac was observed in Ifng promoter, gene body and the conserved noncoding sequence 22 kilobase pairs upstream of Ifng (CNS-22) in KO T cells (
To determine whether reduced cytosolic acetyl-CoA was sufficient to repress IFN-γ expression, we inhibited ATP-citrate lyase (ACL), the enzyme converting citrate to acetyl-CoA, and found that IFN-γ expression was diminished in WT T cells (
To test whether LDHA activity is required for its regulation of cytokine production, a LDHA inhibitor FX11 was used. We found that FX11 dose-dependently inhibited IFN-γ expression in T cells (
In the absence of endogenous 3′UTR, the Ifng transcript in Yeti/Yeti mice is stable, resulting in sustained IFN-γ production and a lethal autoinflammatory phenotype (25). Indeed, all Yeti/Yeti mice succumbed to death by 3 weeks of age (
To further explore aerobic glycolysis in control of effector T cell responses, we used Scurfy mice with a mutation in the Treg cell lineage Foxp3 gene (Foxp3sf) (26). LDHA deficiency in T cells corrected the Scurfy phenotype and extended life span of Foxp3sf mice (
Naïve CD4 T cells (2.5×104) were isolated from spleen and peripheral lymph nodes of Ldhafl/fl (WT) and CD4CreLdhafl/fl (KO) mice. T cells were cultured with antigen-presenting cells (APCs) at a ratio of 10:1 (APC:T) in T-cell medium (RPMI-1640, 10% fetal bovine serum, 2 mM L-glutamine, 55 μM 2-mercaptoethanol, 100 U/mL penicillin, and 100 mg/mL streptomycin) under Th17 differentiation conditions (1 μg/mL anti-CD3, 1 μg/mL anti-CD28, 2 ng/mL TGF-β, 10 ng/mL IL-6, and 10 μg/L α-IL2). WT cells were treated with 0.3 μM, 10 μM, or 30 μM GNE-140, or were untreated. Cultured cells were harvested on day 4, and were stimulated with ionomycin and PMA for 4 hours. Cytokines and transcription factors were analyzed by flow cytometry.
Under untreated conditions, KO T cells produced a significantly lower amount of IL-17A than WT T cells (
The same mechanism that we proposed for LDHA regulation of Th1 cell differentiation also operates in Th17 cells. We found that LDHA-deficiency resulted in diminished IL-17a production under Th17 culture conditions (
To test whether reduced acetyl-CoA production was sufficient to blunt Th17 response, we added an ACL inhibitor (BMS 303141) to the Th17 culture, and found that it repressed IL-17a production (
Overall, our findings in Examples 1-6 do not support a translational mechanism of aerobic glycolysis in IFN-γ production (12), as LDHA promotes IFN-γ expression independent of its 3′UTR. Previous studies have shown that utilization of glycolytic intermediates for biosynthesis accounts for a small fraction (˜7%) of the glucose consumed in activated T cells (27), while aerobic glycolysis produces the majority (˜60%) of ATP (28). Hence, LDHA-mediated aerobic glycolysis may primarily relieve the burden of mitochondria as an energy house to ‘burn’ carbons to generate ATP. As a result, more citrate can be exported out of mitochondria to generate acetyl-CoA and promote histone acetylation in selected gene loci (
Mice with one targeted allele of Ldha (Ldha (Ldhstm1a(EUCOMM)Wtsi) were obtained from EUCOMM, in which the third exon was flanked by two loxp sites. The mice were first crossed with a transgenic Flipase strain (Jackson Laboratory) to remove the Neo cassette, and then crossed with CD4Cre transgenic mice to specifically delete Ldha in T cells. Yeti mice were kindly provided by Dr. Richard Locksley (UCSF). C57BL/6, CD4Cre and Foxp3sf mice were purchased from the Jackson Laboratory. All mice were maintained in the MSKCC animal facility under SPF conditions, and animal experimentation was conducted in accordance with institutional guidelines.
Anti-LDHA (2012S), anti-GAPDH (D16H11), anti-Histone 3 (D2B12), and normal rabbit IgG were purchased from Cell Signaling. Anti-LDHB (60H11), anti-Histone H3 (acetyl K9) (ab4441), anti-Histone H3 (acetyl K27) (ab4729), and anti-RNA polymerase II CTD repeat YSPTSPS (ab5131) were obtained from Abcam. Fluorescent-dye-labeled antibodies against CD4, CD8, TCR-β, CD44, CD62L, CD69, CD25, CD40L, IFN-γ, NK1.1, Foxp3, and T-bet were purchased from eBiosciences, BioLegend, or Tonbo. Blocking antibodies for FcγR (2.4G2) and IL-4 (11B11), as well as anti-CD3 (145-2C11) and anti-CD28 (37.51) were obtained from Bio X Cell.
Cells from thymi, spleens, and lymph nodes were depleted of erythrocytes by hypotonic lysis. Cells were incubated with specific antibodies for 15 min on ice in the presence of anti-FcγR to block FcγR binding. Dead cells were excluded by DAPI (Invitrogen) staining. To determine cytokine expression, cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma) and 1 μM ionomycin (Sigma) in the presence of GolgiStop (BD Biosciences) for 4 h. After stimulation, cells were stained with cell-surface marker antibodies and LIVE/DEAD Fixable dye (Invitrogen) to exclude dead cells, fixed, and permeabilized with a transcription factor staining kit (eBioscience), followed by staining with cytokine antibodies. All samples were acquired with an LSR II flow cytometer (Becton, Dickinson) and analyzed with FlowJo software (TreeStar).
CD62L+CD44−CD25−CD4+ T cells were isolated from spleen and peripheral lymph nodes of wild-type (WT) or CD4CreLdhafl/fl (KO) mice with naïve CD4+ T cells isolation kits (Miltenyi or Stem Cell). Purity was checked by flow cytometry, and was over 98%. For in vitro T cell activation, 0.2-0.5×106 naïve CD4+ T cells were cultured on a 24-well plate pre-coated with 5 μg/mL anti-CD3 in T cell medium [RPMI/1640, 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, lx non-essential amino acid (Invitrogen), 10 mM HEPES, 55 μM 2-mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin] supplemented with 2 μg/mL anti-CD28 and 100 U/ml IL-2. For T helper 1 (Th1) cell differentiation, T cells were cultured with the addition of 10 μg/mL anti-IL-4 and 10 ng/mL IL-12. For T helper 17 (Th17) cell differentiation, T cells were cultured with the addition of 10 μg/mL anti-IL-4, 10 μg/mL anti-IFN-γ, 10 μg/mL anti-IL-2, 2 ng/ml TGF-β, 10 ng/ml IL-6, 10 ng/ml IL-23, and 10 ng/mL IL-1β. For cell proliferation experiments, naïve CD4+ T cells were labeled with CFSE (Invitrogen), and activated on a 24-well plate, pre-coated with 5 μg/mL anti-CD3, in T cell culture medium supplemented with 2 μg/mL anti-CD28 and 100 U/mL IL-2. CFSE dilution was assessed by flow cytometry on days 1, 2, and 3 post-activation. For sodium acetate and TSA treatment experiment, cells were differentiated under Th1 conditions for 3 days, and cultured in T cell medium supplemented with 100 U/mL IL-2, 10 μg/mL anti-IL-4, 10 ng/mL IL-12, and the indicated concentrations of sodium acetate (Sigma), TSA (Promega), or vesicle for 24 h. For ATP citrate lyase (ACL) inhibitor experiments, naïve CD4+ T cells isolated from WT mice were cultured under Th1 conditions for 2 days or under Th17 conditions for 3 days. DMSO and indicated concentrations of ATP citrate lyase inhibitor (BMS-303141, Sigma) were added into the culture for another 24 hours. For LDHA inhibitor experiments, naïve CD4+ T cells isolated from WT mice were cultured under Th1 or Th17 conditions for 3-4 days. DMSO and indicated concentrations of FX11 or GNE-140 were added into the culture for another 24 hours.
3′UTR vectors were co-transfected with pCL-Eco helper plasmid into Phoenix cells, and the culture supernatant was used to infect activated T cells. Naïve CD4+ T cells isolated from WT and KO mice were activated with plated-bound anti-CD3 and soluble anti-CD28 for 2 days, and transduced with retroviral supernatants via spin-infection (2600 rpm for 2 h at 35° C.) and cultured for another 2 days. The GFP signal was examined by flow cytometry.
Heart and muscle tissues were harvested from mice perfused with ice-cold PBS, and homogenized to single cells with non-denaturing cell lysis buffer (Cell Signaling). Activated CD4+ T cells and tissues were lysed with non-denaturing cell lysis buffer on ice for 20 min, and the cell lysate was centrifuged at 20,000 g for 10 min at 4° C. The supernatants were diluted with 2× native loading buffer (Biorad), and subjected to electrophoresis with precast 4-20% native gels (Biorad). The gels were subsequently incubated in a development solution containing 100 mM Tris-HCl (pH 8.0), 0.3 mM NAD (Sigma), 0.1 M sodium lactate (Sigma), 0.5 mM phenazin-methosulphate (Sigma) and 0.4 mM tetrazolium-blue (Sigma) at 37° C. for 30 min.
Cells were lysed with a cell lysis buffer (Cell Signaling), and protein concentrations were determined with a BCA kit (Thermo Scientific). Protein extracts were separated by SDS-PAGE gels and transferred to nitrocellulose blotting membranes (GE Healthcare). The membranes were probed with antibodies and visualized with the Immobilon Western Chemiluminescent HRP Substrate (Millipore).
To determine glucose consumption and lactate production of activated CD4+ T cells, culture medium was replaced with fresh medium 48 h after T cell activation, and collected 24 h later. Medium alone in wells of the same plate was used as control. Glucose and lactate levels in culture media were measured with a glucose assay kit II (BioVison) and lactate assay kit II (Sigma).
For measurement of cytosolic acetyl-CoA, naïve CD4+ T cells were differentiated under Th1 conditions for 2 days and then cultured in fresh T cell medium for another 24 h. Cells were lysed with lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH=7.4, 150 mM NaCl) on ice for 10 min. The lysates were spun at 20,000 g for 10 min at 4° C., the pellets (nuclei and heavy membrane) were discarded, and the supernatants were used for acetyl-CoA measurement with an Acetyl-CoA Assay Kit (Sigma). Freshly isolated naïve WT and KO CD4+ T cells were rested in T cell medium with 10 ng/mL IL-7 for 4 h at 37° C. in the incubator, and the level of acetyl-CoA was measured.
OCR and ECAR were measured with an XF96 extracellular flux analyzer (Seahorse Bioscience), following protocols recommended by the manufacturer. Briefly, freshly isolated CD4+ naïve or activated cells were seeded on XF96 microplates (150,000 cells/well) that had been pre-coated with Cell-Tak adhesive (BD Biosciences). The plates were quickly centrifuged to immobilize cells. Cells were maintained in a non-buffered assay medium (Seahorse Biosciences) in a non-CO2 incubator for 30 min before the assay. Glycolysis was measured with XF glycolysis stress test kit (Seahorse Biosciences). Initially, cells were incubated in the glycolysis stress test medium without glucose, and ECAR was assessed. Three baseline recordings were made, followed by sequential injection of 10 mM glucose and 1 μM oligomycin, which inhibited mitochondrial ATP production and shifted the energy production to glycolysis. The increased ECAR revealed the maximum glycolytic capacity of T cells. The final injection was 100 mM 2-DG, a glucose analog that inhibited glycolysis. The resulting decrease of ECAR confirmed that the ECAR observed in experiments was due to glycolysis. The Mito stress test kit (Seahorse Biosciences) was used to test OCR under different conditions. Three baseline recordings were made, followed by sequential injection of 1 μM oligomycin, 0.25 μM FCCP, which uncoupled oxygen consumption from ATP production to obtain maximal OCR, and 0.5 μM rotenone/antimycin A, which inhibited complex I and III.
Metabolic flux analysis of 13C6-glucose in Th1 cells was determined by CE-MS. Briefly, ˜10 million WT or KO Th1 cells were labeled with 13C6-glucose (10 mM) in glucose-deficient RPMI1640 supplemented with 10% dialyzed FBS and 100 U/mL IL-2 for 2 h. Metabolites were extracted according to the Floating Cells Protocol E-150782, and shipped to Human Metabolome Technologies Inc. (Boston) for CE-MS analysis (F-SCOPE). Data were from three biological replicates.
Total RNA was extracted with RNeasy kit (QIAGEN), and cDNA libraries were generated and sequenced using a HiSeq 2000 platform (Illumina) at the Integrated Genomics Operation of MSKCC. For quantitative RT-PCR, RNA was reverse transcribed with Superscript III (Invitrogen). mRNA levels were normalized to β-Actin. The primers used were: Actin-F: 5′-GGCACCACACCTTCTACAATG-3′ (SEQ ID NO: 1); Actin-R: 5′-GTGGTGGTGAAGCTGTAGCC-3′ (SEQ ID NO: 2). Ifng-F: 5′-CTTTGGACCCTCTGACTTGAG-3′ (SEQ ID NO: 3), Ifng-R: 5′-TTCCACATCTATGCCACTTGAG-3′ (SEQ ID NO: 4).
Quality Control of raw reads was done using FastQC (v0.11.2) (10) to ensure that there were no major flaws in sequencing. They were then mapped to mm10 genome using STAR (v2.3.0e_r291) (30) and default parameters. The mapped reads were counted using htseq-count (v0.6.0, parameters −t exon) (31) and gene models from Ensembl (Mus_musculus.GRCm38.75.gtf). Differential expression was performed using DESeq2 (v1.2.10, default parameters) (32). Human orthologs were retrieved from Ensembl v75. DESeq2 normalized counts with human orthologs were provided to GSEA (v2-2.0.14) (33) for gene set enrichment analysis.
Chromatin immunoprecipitations were performed with SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling) according to manufacturer'"'"'s instructions. Briefly, naïve CD4+ T cells were differentiated to Th1 cells for 3 days, and were fixed for 10 min at 25° C. with 1% formaldehyde. After incubation, glycine was added to a final concentration of 0.125 M to quench formaldehyde. Subsequently, cells were lysed, and chromatin was harvested and fragmented using enzymatic digestion, followed by sonication. The chromatin was then subjected to immunoprecipitation with anti-H3K9Ac at 4° C. overnight, and was incubated with protein G magnetic beads at 4° C. for 2 h. The immune complexes were washed and eluted in 150 μL elution buffer. Elute DNA and input DNA were incubated at 65° C. to reverse the crosslinking. After digestion with proteinase K, DNA was purified with spin columns. The library was prepared with KAPA library preparation kit (Kapa Biosystems) and sequenced using a HiSeq 2000 platform (Illumina) at the Integrated Genomics Operation of MSKCC. The relative abundance of precipitated DNA fragments was analyzed by qPCR using Power SYBR Green PCR Master Mix (Qiagen) and the enrichments were normalized to Cd3e promoter. The following primers were used for qPCR: Cd3e promoter-F: 5′-TCAGTGTGGAGGTGCTTTG-3′ (SEQ ID NO: 5), Cd3e promoter-R: 5′-CAGCCTTCCCATAAGGATGAA-3′ (SEQ ID NO: 6); Ifng promoter-F: 5′-GGAGCCTTCGATCAGGTATAAA-3′ (SEQ ID NO: 7), Ifng promoter-R: 5′-CTCAAGTCAGAGGGTCCAAAG-3′ (SEQ ID NO: 8); CNS22-F: 5′-GAGGCCAAATTTCTGCTCATTG-3′ (SEQ ID NO: 9), CNS22-R: 5′-GTTCTTTCAGGAAGCCCGTTA-3′ (SEQ ID NO: 10).
Reads were first trimmed using Trimmomatic (v0.33) (34). Reads were trimmed if first/last 3 nucleotides had phred quality score of <15, or at the point where a sliding window of 4 nucleotides averaged a phred quality score <15. Illumina adapters were also removed. The reads were then aligned using bowtie2 (v 2.2.6, options −fr—no-discordant) (35). Multimapping reads were removed after alignment. Peak calling was done using MACS2 (v2.1.0) (36) on pooled replicates and individual samples using p-value cutoff of 0.01. The peaks were then filtered further using IDR (37) to make sure the peaks were consistent among replicates. The promoter was annotated as region within 2,000 bp from TSS, intron was annotated as region within the gene body but not in the promoter region, and peaks at regions outside but close to gene body were annotated as intergenic.
RNA-seq and ChIP-Seq data are deposited in the Genome Expression Omnibus under accession number GSE86188, the content of which is incorporated herein by reference.
Tissues from euthanized animals were fixed in Safefix II (Fisher) and embedded in paraffin. 5-μm sections were stained with haematoxylin and eosin.
Statistical tests were performed with Prism (GraphPad). A value of p<0.05 were considered statistically significant. All error bars represent standard error (SD).
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The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. The present invention is further described by the following claims.