Summary of Study ST004334
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 PR002747. The data can be accessed directly via it's Project DOI: 10.21228/M8KG2R 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 | ST004334 |
| Study Title | Oxidative pentose phosphate pathway is required for T cell activation and anti-tumor immunity - PGD-knockout |
| Study Summary | To strengthen our finding in G6PD knockout, we performed metabolomics to evaluate dysregulated metabolic pathways in activated PGD-knockout CD8 T cells as supporting evidence. Similar to G6PD knockout, PGD knockout upregulated SLC7A11 (cystine-glutamate antiporter), leading to cystine accumulation and depletion of glutathione and glutathione disulfide, albeit to a lesser degree. Distinctly, PGD knockout accumulated 6-phosphogluconate and its dephosphorylated product gluconate, consistent with continued G6PD activity but blockade at the downstream PGD step. |
| Institute | Princeton University |
| Laboratory | Carl Icahn Laboratory |
| Last Name | Chen |
| First Name | Zihong |
| Address | Carl Icahn Laboratory, South Drive, Princeton, New Jersey, 08544, USA |
| zihongc@princeton.edu | |
| Phone | 6099333507 |
| Submit Date | 2025-10-04 |
| Study Comments | Study part 2 of 2 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-11-28 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002747 |
| Project DOI: | doi: 10.21228/M8KG2R |
| Project Title: | Oxidative pentose phosphate pathway is required for T cell activation and anti-tumor immunity |
| Project Summary: | Glucose is catabolized by two major metabolic pathways, glycolysis and the oxidative pentose phosphate pathway (oxPPP). The oxPPP generates NADPH at two steps, glucose-6-phosphate dehydrogenase (G6PD), the most common enzyme deficiency in humans, and 6-phosphogluconate dehydrogenase (PGD). Previous literature suggests that G6PD supports but PGD limits T cell-mediated immunity. Here we use T cell-specific knockout mouse models to show that both enzymes are required for anti-tumor immunity and response to immunotherapy. PGD knockout depletes mature T cells systemically, while G6PD loss does not reduce basal T cell populations but results in apoptosis upon activation. Such apoptosis is not reversed by major downstream products of the oxPPP, including antioxidants, nucleosides, or fatty acids. Instead, T cells are partially rescued by removal of media cystine, whose reduction requires NADPH. G6PD loss induces an oxidative stress response that upregulates cystine import, which together with low NADPH leads to fatal disulfide stress. Overall, these results highlight an essential role for the oxidative pentose phosphate pathway in cystine homeostasis and T cell-mediated immunity. |
| Institute: | Princeton University |
| Last Name: | Chen |
| First Name: | Zihong |
| Address: | Carl Icahn Laboratory, South Drive, Princeton, New Jersey, 08544, USA |
| Email: | zihongc@princeton.edu |
| Phone: | 6099333507 |
Subject:
| Subject ID: | SU004493 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6NTac |
| Age Or Age Range: | 7-8 months old |
| Gender: | Female |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Sample source |
|---|---|---|---|
| SA509129 | blank | NA | blank |
| SA509130 | ctrl-1 | Pgd ctrl | splenic CD8+ T cells |
| SA509131 | ctrl-2 | Pgd ctrl | splenic CD8+ T cells |
| SA509132 | ctrl-3 | Pgd ctrl | splenic CD8+ T cells |
| SA509133 | ctrl-unlabeled | Pgd ctrl | splenic CD8+ T cells |
| SA509134 | ko-1 | Pgd ko | splenic CD8+ T cells |
| SA509135 | ko-2 | Pgd ko | splenic CD8+ T cells |
| SA509136 | ko-3 | Pgd ko | splenic CD8+ T cells |
| Showing results 1 to 8 of 8 |
Collection:
| Collection ID: | CO004486 |
| Collection Summary: | To isolate naïve CD8+ T cells, mouse spleens were collected and pooled as single-cell suspensions by manual disruption with a syringe plunger and passage through 40 µm cell strainers (229482, Celltreat) into RPMI-1640 medium (11875-093, Gibco). After RBC lysis (00-4300-54, Invitrogen), splenocytes were subjected to magnetic bead separation using naïve CD8a+ T Cell Isolation Kit, mouse (130-096-543, Miltenyi Biotec Inc.) following vendor instructions. Naïve T cells were cultured at 1 × 10^6 cells ml-1 in complete RPMI medium (RPMI-1640 medium, 10% heat inactivated complete FBS (35-011-CV, Corning), 100 U ml−1 penicillin, 100 µg ml−1 streptomycin (15140-122, Gibco) and 55 μM 2-mercaptoethanol (21985-023, Gibco)) and activated with plate-bound anti-CD3 (10 µg ml−1; BE0001-1, Bio X Cell), anti-CD28 (5 µg ml−1; BE0015-1, Bio X Cell) and soluble recombinant murine IL-2 (20 ng ml−1; 212-12, Peprotech) for 24 h. While irrelevant for metabolomics, for the last 2 h, tracing medium containing 100% [1,2-13C] glucose (CLM-504, Cambridge Isotope Laboratories, Inc.) was spiked in to achieve 20% glucose labeling. |
| Sample Type: | T-cells |
Treatment:
| Treatment ID: | TR004502 |
| Treatment Summary: | T cells were harvested from mice with the following genotypes: Pgd ctrl: Pgd flox/flox; Pgd ko: Pgd flox/flox, CD4-Cre. |
Sample Preparation:
| Sampleprep ID: | SP004499 |
| Sampleprep Summary: | 1 × 10^6 activated T cells were rapidly pelleted (6,000 rpm, 30 s, room temperature). The medium was quickly removed, followed by immediate addition of 70 μl of ice-cold acetonitrile: methanol: water (2:2:1, supplemented with 0.5 vol% formic acid which increases triphosphate yield). Extracts were mixed by pipetting, kept on ice, and neutralized by NH4HCO3 (15% in water, 6.1 µL) five minutes after extraction. Neutralized extracts were kept on dry ice for 1h and centrifuged (21,300 × g, 30 min at 4°C). Supernatants were used for LC-MS analysis. Natural isotope abundance was corrected by the ‘accucor’ package on R. |
Chromatography:
| Chromatography ID: | CH005495 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters XBridge BEH amide (150 x 2.1 mm, 2.5um) |
| Column Temperature: | 25 |
| Flow Gradient: | 0-2 min, 90% B; 3-7 min, 75% B; 8-9 min, 70% B; 10-12 min, 50% B; 12-14 min, 25% B; 16-20.5 min, 0.5% B; 21-25 min, 90% B. |
| Flow Rate: | 0.15 mL/min |
| Solvent A: | 95% water/5% acetonitrile; 20 mM ammonium acetate; 20 mM ammonium hydroxide |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
Analysis:
| Analysis ID: | AN007237 |
| Analysis Type: | MS |
| Chromatography ID: | CH005495 |
| Num Factors: | 3 |
| Num Metabolites: | 122 |
| Units: | Arbitrary Units |
| Analysis ID: | AN007238 |
| Analysis Type: | MS |
| Chromatography ID: | CH005495 |
| Num Factors: | 3 |
| Num Metabolites: | 237 |
| Units: | Arbitrary Units |
