Summary of Study ST003167
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 | ST003167 |
| Study Title | Metabolomics of Human CD19-BBz CD8 CAR-T cells with low and high MCJ expression |
| Study Summary | The goal of these experiments are to characterize the metabolic profile of the human CD8 CD19-BBz CAR-T cells from donors with high or low MCJ expression after 3 expansions (6 days) with IL-2 (100IU/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: | SU003286 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Factor | Sample source |
|---|---|---|---|
| SA343035 | D26 19BBz_4 | high 19BBz | T-Cells |
| SA343036 | D26 19BBz_3 | high 19BBz | T-Cells |
| SA343037 | D26 19BBz_2 | high 19BBz | T-Cells |
| SA343038 | D16 19BBz_3 | low 19BBz | T-Cells |
| SA343039 | D16 19BBz_4 | low 19BBz | T-Cells |
| SA343040 | D16 19BBz_2 | low 19BBz | T-Cells |
| Showing results 1 to 6 of 6 |
Collection:
| Collection ID: | CO003279 |
| Collection Summary: | Human CD8 cells were isolated with positive selection as described. The CD8 cells were activated with anti-CD3/anti-CD28 beads (Dynabeads™ Human T-Expander CD3/CD28, Gibco), as previously described107. After 48h activation, the CD8 cells were spun (1000xg for 2h at 32C) with lentiviral supernatant containing CD19-BBz/shMCJ-1 CAR, CD19-BBz/shMCJ-2 CAR or CD19-BBz/shMCJ-2 CAR construct-packing virus with rhIL-2 (40 IU/ml) and protamine sulfate. After transduction, the anti-CD3/anti-CD28 beads were removed and CD8 cells were expanded with rhIL-2 (100 IU/ml) for the specified number of days. |
| Sample Type: | T-cells |
Treatment:
| Treatment ID: | TR003295 |
| Treatment Summary: | The human CD19-BBz shRNA CAR lentiviral constructs were based on a previously described CD19-BBz CAR containing the human CD19-binding scFV FMC63, CD8 hinge domain, 4-1BB costimulatory domain and CD3 chain105. Using CD19-BBz CAR plasmid as a cloning vector, we generated multiple vectors where we incorporated the RNA polymerase III U6 promotor (on the 3' of the CD3 chain domain) followed by an shRNA: 1) a CD19-BBz/shMCJ-1 CAR construct containing the shMCJ-1 5’-GAAGATTTCAACTCCTAGC-3’ sequence106, 2) a CD19-BBz/shMCJ-2 CAR construct containing the shMCJ-2; 5’-AACCTCTAGAACAAGTTATC-3’, and 3) a CD19-BBz/c-shRNA CAR vectors expressing the shRNA encoding scramble sequences. Lentiviral supernatant was produced in the LentiX-293T packaging cell line (Clonetech) as previously described. Lentiviral supernatants were collected after 48 hours post-transfection. |
Sample Preparation:
| Sampleprep ID: | SP003293 |
| 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 | AN005196 | AN005197 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Exploris120 | Exploris120 |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | Peak Areas | Peak Areas |
Chromatography:
| Chromatography ID: | CH003931 |
| Chromatography Summary: | Negative ion Mode |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-0.2 min 5% B, 0.2-.8 min hold at 95% B, .8-.81 min 95-5% B, .81-1 min hold at 5% B. |
| Flow Rate: | 0.450 ml/min |
| Solvent A: | 100% Water; 10mM Ammonium acetate, .01% Formic Acid |
| Solvent B: | 50% Methanol/ 50% Acetonitrile; 10mM Ammonium acetate, .01% Formic Acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003932 |
| Chromatography Summary: | Positive Ion Mode |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-0.3 min 5% B, 0.3-.8 min hold at 95% B, .8-.81 min 95-5% B, .81-1 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: | MS004929 |
| Analysis ID: | AN005196 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Exploris120 |
| 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: | MS004930 |
| Analysis ID: | AN005197 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Exploris120 |
| 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 |
