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.
| Study ID | ST001373 |
| Study Title | Targeting Sirt2 reprograms T cell metabolism for effective immune response |
| Study Type | Targeted Metabolomics |
| Study 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 |
| john.koomen@moffitt.org | |
| Phone | 8137458524 |
| Submit Date | 2019-07-24 |
| Num Groups | 2 |
| Total Subjects | 9 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-01-25 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
|---|---|---|
| SA099875 | Sirt2KO2 | Sirt2 Knockout |
| SA099876 | Sirt2KO3 | Sirt2 Knockout |
| SA099877 | Sirt2KO4 | Sirt2 Knockout |
| SA099878 | Sirt2KO1 | Sirt2 Knockout |
| SA099879 | WT5 | Wild-type |
| SA099880 | WT2 | Wild-type |
| SA099881 | WT3 | Wild-type |
| SA099882 | WT4 | Wild-type |
| SA099883 | WT1 | Wild-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 -80C for at least one hour. After addition of the extraction solvent, the samples are then placed in the -80C 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 -80C 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: | 100% water; 10 mM ammonium carbonate; 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 |
