Summary of study ST001373

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000939. The data can be accessed directly via it's Project DOI: 10.21228/M89T1B This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001373
Study TitleTargeting Sirt2 reprograms T cell metabolism for effective immune response
Study TypeTargeted Metabolomics
Study SummaryThere is a growing evidence that metabolism is a key driver of T cell functions. A switch from oxidative phosphorylation to aerobic glycolysis is a hallmark of T cell activation and is required to meet metabolic demands of proliferation and effector functions. However the mechanisms underlying the metabolic switch in T cells remain unclear. Here we identify Sirt2 as a crucial immune checkpoint coordinating metabolic and functional fitness of T cells. Sirt2 is induced upon T cells activation and increases in late maturation stages. Sirt2 negatively regulates glycolysis by targeting key glycolytic enzymes. Remarkably, Sirt2 knockout T cells exhibit profound upregulation of aerobic glycolysis with enhanced proliferation and effector function and thus effectively reject tumor challenge in vivo. Furthermore pharmacologic inhibition of Sirt2 in human tumor infiltrating lymphocytes demonstrated similar phenotype. Taken together our results demonstrate Sirt2 as an actionable target to reprogram T cell metabolism to augment immunotherapy.
Institute
Moffitt Cancer Center
DepartmentImmunology
LaboratorySungjune Kim
Last NameKoomen
First NameJohn
Address12902 Magnolia Drive
Emailjohn.koomen@moffitt.org
Phone8137458524
Submit Date2019-07-24
Num Groups2
Total Subjects9
Raw Data AvailableYes
Analysis Type DetailLC-MS
Release Date2021-01-25
Release Version1
John Koomen John Koomen
https://dx.doi.org/10.21228/M89T1B
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000939
Project DOI:doi: 10.21228/M89T1B
Project Title:Targeting Sirt2 reprograms T cell metabolism for effective immune response
Project Type:Targeted Metabolomics
Project Summary:There is a growing evidence that metabolism is a key driver of T cell functions. A switch from oxidative phosphorylation to aerobic glycolysis is a hallmark of T cell activation and is required to meet metabolic demands of proliferation and effector functions. However the mechanisms underlying the metabolic switch in T cells remain unclear. Here we identify Sirt2 as a crucial immune checkpoint coordinating metabolic and functional fitness of T cells. Sirt2 is induced upon T cells activation and increases in late maturation stages. Sirt2 negatively regulates glycolysis by targeting key glycolytic enzymes. Remarkably, Sirt2 knockout T cells exhibit profound upregulation of aerobic glycolysis with enhanced proliferation and effector function and thus effectively reject tumor challenge in vivo. Furthermore pharmacologic inhibition of Sirt2 in human tumor infiltrating lymphocytes demonstrated similar phenotype. Taken together our results demonstrate Sirt2 as an actionable target to reprogram T cell metabolism to augment immunotherapy.
Institute:Moffitt Cancer Center
Department:Immunology
Laboratory:Sungjune Kim
Last Name:Koomen
First Name:John
Address:12902 Magnolia Drive
Email:john.koomen@moffitt.org
Phone:8137458524
Contributors:Imene Hamaidi1, Lin Zhang2, Nayoung Kim3, Min Hsuan Wang1, Cristina Iclozan1, Bin Fang4, Min Liu4, John Koomen4, Anders Berglund5, Sungjune Kim1,6*

Subject:

Subject ID:SU001447
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype
SA099875Sirt2KO2Sirt2 Knockout
SA099876Sirt2KO3Sirt2 Knockout
SA099877Sirt2KO4Sirt2 Knockout
SA099878Sirt2KO1Sirt2 Knockout
SA099879WT5Wild-type
SA099880WT2Wild-type
SA099881WT3Wild-type
SA099882WT4Wild-type
SA099883WT1Wild-type
Showing results 1 to 9 of 9

Collection:

Collection ID:CO001442
Collection Summary:CD8+ purified T cells from WT (n = 5) and Sirt2 KO (n = 4) mouse spleens were stimulated with plate-coated anti-CD3 (5 μg/ml, BXCELL) for 72h, followed by extensive washing of the cell pellets with PBS. All processes are carried out on ice. An aliquot (1 mL) of 80% MeOH extraction solvent is added to the sample (with Internal Standards spiked in) for protein precipitation. The 80% MeOH extraction solvent is is precooled to -80C for at least one hour. After addition of the extraction solvent, the samples are then placed in the -80C freezer for 30 minutes. After incubation, the samples are immediately centrifuged at 18,800 × g (Microfuge 22R, Beckman Coulter) at 4 C for 10 minutes. After centrifugation, the supernatant is transferred to new a microcentrifuge tube (Eppendorf brand) for drying by vacuum concentration. The dried metabolites are re-dissolved in 80% MeOH. The precipitated protein pellet is resolubilized for Bradford assays to measure the protein concentration as a quality control for the sample loading.
Sample Type:Spleen
Collection Method:T-cell isolation
Collection Location:Moffitt Cancer Center
Collection Frequency:1 time
Volumeoramount Collected:2,000,000 cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001462
Treatment Summary:CD8+ purified T cells from WT (n = 5) and Sirt2 KO (n = 4) mouse spleens were stimulated with plate-coated anti-CD3 (5 μg/ml, BXCELL) for 72h, followed by extensive washing of the cell pellets with PBS.
Treatment:anti-CD3 stimulation
Treatment Compound:antibody
Treatment Route:Cells placed on coated plate
Treatment Doseduration:72 hours

Sample Preparation:

Sampleprep ID:SP001455
Sampleprep Summary:All processes are carried out on ice. An aliquot (1 mL) of 80% MeOH extraction solvent is added to the sample (with Internal Standards spiked in) for protein precipitation. The 80% MeOH extraction solvent is precooled to -80C for at least one hour. After addition of the extraction solvent, the samples are then placed in the -80 oC freezer for 30 minutes. After incubation, the samples are immediately centrifuged at 18,800 × g (Microfuge 22R, Beckman Coulter) at 4 oC for 10 minutes. After centrifugation, the supernatant is transferred to new a microcentrifuge tube (Eppendorf brand) for drying by vacuum concentration. The dried metabolites are re-dissolved in 80% MeOH. The precipitated protein pellet is resolubilized for Bradford assays to measure the protein concentration as a quality control for the sample loading.
Processing Storage Conditions:On ice
Extraction Method:Methanol
Extract Enrichment:Vacuum centrifugation
Sample Resuspension:80% MeOH
Sample Derivatization:None
Sample Spiking:Internal Standards added
Subcellular Location:N/A

Combined analysis:

Analysis ID AN002293
Analysis type MS
Chromatography type HILIC
Chromatography system Vanquish
Column SeQuant ZIC-pHILIC
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap
Ion Mode NEGATIVE
Units Peak Intensity

Chromatography:

Chromatography ID:CH001684
Chromatography Summary:UHPLC-MS was performed using a Vanquish LC (Thermo, San Jose, CA) interfaced with a Q Exactive HF mass spectrometer (Thermo, San Jose, CA). Chromatographic separation was performed on a SeQuant ZIC-pHILIC LC column (150 × 4.6 mm, 5 µm particle size, MilliporeSigma, Burlington, MA). In order to maintain the stable column pressure and further filter the LC solvents, a SeQuant ZIC-pHILIC guard column (20 × 4.6 mm, 5 µm particle size, MilliporeSigma, Burlington, MA) is connected before the LC column. The mobile phase A was aqueous 10 mM ammonium carbonate and 0.05% ammonium hydroxide, and the mobile phase B was 100% acetonitrile. The total running time is 20 minutes. The column temperature was set to 30°C, and the injection volume is 2 µL.
Instrument Name:Vanquish
Column Name:SeQuant ZIC-pHILIC
Column Temperature:30
Flow Gradient:80% B to 20%B over 13 minutes
Flow Rate:0.25 ml/min
Injection Temperature:5
Internal Standard:D-Glucose (2,3,4,5,6-13C5), D-Glucose-6-phosphate (U-13C6), D-Fructose-1, 6-bisphosphate (U-13C6), L-Serine (13C3), Glycine (1,2-13C2), L-Cysteine (3,3-D2), Phosphoenol Pyruvate (2,3-13C2), Lactate (3,3,3-D3), Pyruvate (D3), Acetyl-1,2-13C2 CoA, Citric Acid (2,2,4,4-D4), Alpha-Ketoglutaric Acid (1,2,3,4-13C4), Succinic Acid (D4), Fumaric Acid (D4), DL-Malic Acid (2,3,3-D3), D-Fructose-6-phosphate (U-13C6) are all from Cambridge Isotope Labs.
Solvent A:aqueous 10 mM ammonium carbonate and 0.05% ammonium hydroxide
Solvent B:100% acetonitrile
Analytical Time:20
Chromatography Type:HILIC

MS:

MS ID:MS002137
Analysis ID:AN002293
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Full MS is performed in positive and negative mode separately, and the mass scan range is 60 to 900 m/z. In addition to MS data acquisition 1, parallel reaction monitoring (PRM) data are acquired for the important intermediates involved in Glycolysis and the TCA cycle.
Ion Mode:NEGATIVE
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