Summary of Study ST003166
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 PR001970. The data can be accessed directly via it's Project DOI: 10.21228/M8314P 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 | ST003166 |
| Study Title | Metabolomics of Murine WT or MCJ KO CD19-BBz CD8 CAR-T cells |
| Study Summary | MCJ/DnaJC15 is an endogenous negative regulator of Complex I and mitochondrial respiration. The goal of these experiments are to characterize the metabolic profile of murine WT or MCJ KO CD8 CD19-BBz CAR-T cells after 3 expansions (6 days) with IL-2 (60IU/ml). |
| Institute | University of Colorado School of Medicine |
| Laboratory | Laboratory of Angelo D'Alessandro in collaboration with Mercedes Rincon |
| Last Name | Cendali |
| First Name | Francesca |
| Address | 13199 East Montview Boulevard, Aurora, CO, 80045, USA |
| francesca.cendali@cuanschutz.edu | |
| Phone | 3037246131 |
| Submit Date | 2024-04-11 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-05-02 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001970 |
| Project DOI: | doi: 10.21228/M8314P |
| Project Title: | Releasing the mitochondrial respiration brake MCJ/DnaJC15 enhances CD8 CAR-T cell therapy efficacy |
| Project Summary: | Metabolism of chimeric antigen receptor (CAR) T cells is emerging as an important area to improve CAR-T cell therapy in cancer treatment. Mitochondrial respiration is essential for survival and function of CAR-T cells, but developing strategies to specifically enhance mitochondrial respiration has been challenging. Here we identify MCJ/DnaJC15, an endogenous negative regulator of mitochondrial Complex I, as a metabolic target to enhance mitochondrial respiration in CD8 CAR-T cells. Loss of MCJ in CD8 CAR-T cells increases their in vitro and in vivo efficacy against mouse B cell leukemias. MCJ deficiency in TCR- specific CD8 cells also increases their efficacy against solid tumors in vivo. Furthermore, we reveal that human CD8 cells express MCJ and that silencing MCJ expression increases mitochondrial metabolism and anti-tumor activity of human CAR-T cells. Thus, targeting MCJ to enhance mitochondrial metabolism is a promising therapeutic strategy to improve the efficacy of adoptive T cell therapies. |
| Institute: | University of Colorado School of Medicine |
| Laboratory: | Laboratory of Angelo D'Alessandro in collaboration with Mercedes Rincon |
| Last Name: | Cendali |
| First Name: | Francesca |
| Address: | 13199 East Montview Boulevard, Aurora, CO, 80045, USA |
| Email: | francesca.cendali@cuanschutz.edu |
| Phone: | 3037246131 |
Subject:
| Subject ID: | SU003285 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | factor | Sample source |
|---|---|---|---|
| SA343027 | MCJ KO CD19-BBz CART_3 | MCJ KO | T-Cells |
| SA343028 | MCJ KO CD19-BBz CART_4 | MCJ KO | T-Cells |
| SA343029 | MCJ KO CD19-BBz CART_2 | MCJ KO | T-Cells |
| SA343030 | MCJ KO CD19-BBz CART_1 | MCJ KO | T-Cells |
| SA343031 | WT CD19-BBz CART_2 | WT | T-Cells |
| SA343032 | WT CD19-BBz CART_3 | WT | T-Cells |
| SA343033 | WT CD19-BBz CART_4 | WT | T-Cells |
| SA343034 | WT CD19-BBz CART_1 | WT | T-Cells |
| Showing results 1 to 8 of 8 |
Collection:
| Collection ID: | CO003278 |
| Collection Summary: | Mouse CD8 cells were isolated with negative selection as described above, activated with anti-CD3/anti-CD28 beads (Dynabeads™ Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation, Gibco, Cat#11453D) with addition of recombinant human IL-2 (rhIL-2) (60 IU/ml) and human recombinant IL-7 (10 ng/ml). On the second and third day of activation, the supernatant containing the CD19-BBz CAR/hEGFR retrovirus was spun on a plate coated with retronectin (Takara Bio Inc.) (2000xg for 2.5h at 32C). The activated cells were added to the viral-coated plate for transduction. The transduced CD8 CAR-T cells were removed from the beads on the fourth day and expanded with rhIL-2 (60 IU/ml) or rhIL-7 (10 ng/ml) and rhL-15 (100 ng/ml). After 2 days (1st expansion) cells were split 1/3 with fresh cytokine-containing medium. After 2 more days (2nd expansion), cells were split again with fresh cytokine-containing medium. Two days later (3rd expansion) cells were used for experiment or incubated in cytokine free-medium. For CAR+ cells enrichment, the CAR-T cells expanded with IL-2 for 3 expansions were incubated with an anti-hEGFR-PE (BioLegend, Cat#352904, RRID: AB_10896794) antibody, followed by incubation with anti-PE microbeads (Miltenyi, order#130-048-801), and purified using the Miltenyi LS columns as recommended by the manufacturer (Miltenyi). The enrichment resulted in a 98-100% CAR+ population, as verified by flow cytometry. |
| Sample Type: | T-cells |
Treatment:
| Treatment ID: | TR003294 |
| Treatment Summary: | Metabolomics differences in this experiment are observed at baseline |
Sample Preparation:
| Sampleprep ID: | SP003292 |
| Sampleprep Summary: | CAR-T cells were isolated as described above either from in vitro culture or from bone marrow harvested in an in vivo study. The cells were washed in PBS and frozen at -80C until the assay is ready to run. Metabolites from cells were extracted at 2x106 cells/ml at 4°C (30 min) in the presence of 5:3:2 MeOH:MeCN:water (v/v/v). The samples were spun down and the resulting supernatant was transferred to new tubes and dried under a vacuum. The resulting residue was reconstituted in 0.1% formic acid at a 3x concentration, then analyzed on a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive MS as previously described in detail. |
Combined analysis:
| Analysis ID | AN005194 | AN005195 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | Peak Area | Peak Area |
Chromatography:
| Chromatography ID: | CH003929 |
| Chromatography Summary: | Negative ion Mode |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B |
| Flow Rate: | 0.450 ml/min |
| Solvent A: | 95% water/5% acetonitrile; 1 mM ammonium acetate |
| Solvent B: | 95% acetonitrile/5% water; 1 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003930 |
| Chromatography Summary: | Positive Ion Mode |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B. |
| Flow Rate: | 0.450 ml/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004927 |
| Analysis ID: | AN005194 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS004928 |
| Analysis ID: | AN005195 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database. |
| Ion Mode: | POSITIVE |
