Summary of Study ST003169

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.

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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 IDST003169
Study TitleMetabolomics of infused Murine WT or MCJ KO CD19-BBz CD8 CAR-T cells from Leukemia-bearing Mice
Study SummaryMCJ/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 in vivo after infused to the leukemia-bearing mice.
Institute
University of Colorado School of Medicine
LaboratoryLaboratory of Angelo D'Alessandro in collaboration with Mercedes Rincon
Last NameCendali
First NameFrancesca
Address13199 East Montview Boulevard, Aurora, CO, 80045, USA
Emailfrancesca.cendali@cuanschutz.edu
Phone3037246131
Submit Date2024-04-11
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-05-02
Release Version1
Francesca Cendali Francesca Cendali
https://dx.doi.org/10.21228/M8314P
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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:SU003288
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
SA3430646MCJ KO CART in vivo T-Cells
SA3430655MCJ KO CART in vivo T-Cells
SA3430664MCJ KO CART in vivo T-Cells
SA3430672WT CART in vivo T-Cells
SA3430683WT CART in vivo T-Cells
SA3430691WT CART in vivo T-Cells
Showing results 1 to 6 of 6

Collection:

Collection ID:CO003281
Collection Summary:The E2a cells (106) were cultured in CMM for no more than 2 passages before intravenous tail vail injection to B6 mice 3 days before CAR-T cell injection. The E2a-bearing mice were irradiated with 500cGy sublethal dosage the day before CAR-T cell injection. The murine CD8 CAR-T cells were generated and expanded with rhIL-2 (60 IU/ml) as described above. The expanded CD8 CAR-T cells were washed with PBS, and 106 CAR+ cells were injected into the E2a-bearing mice via intravenous tail vail injection. The mice were followed for survival with the humane end point of development of hind limb paralysis, or weight loss of more than 20% of the original weight, or signs of major discomfort as determined by the institutional veterinarian.
Sample Type:T-cells

Treatment:

Treatment ID:TR003297
Treatment Summary:Metabolomics differences in this experiment are observed at baseline

Sample Preparation:

Sampleprep ID:SP003295
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 -80C 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 AN005200 AN005201
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Exploris120 Exploris120
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 Areas Peak Areas

Chromatography:

Chromatography ID:CH003934
Chromatography Summary:Negative ion Mode
Instrument Name:Exploris120
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:CH003935
Chromatography Summary:Positive Ion Mode
Instrument Name:Exploris120
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:MS004933
Analysis ID:AN005200
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:MS004934
Analysis ID:AN005201
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
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