Summary of Study ST002084

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 PR001324. The data can be accessed directly via it's Project DOI: 10.21228/M8K989 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	ST002084
Study Title	A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy(Part 2)
Study Summary	Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ T cells, and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knock-in or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression re-shaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together these findings provide a system for identification of GOF immune boosters, and demonstrate PRODH2 as a target to enhance CAR-T efficacy.
Institute	Yale University
Last Name	Ye
First Name	Lupeng
Address	850 West campus drive
Email	lupeng.ye@yale.edu
Phone	2035436568
Submit Date	2022-02-14
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2022-02-28
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001324
Project DOI:	doi: 10.21228/M8K989
Project Title:	Proline metabolism in T cells
Project Summary:	T cell metabolites detection after PRODH2-CD22-CAR knock-in.
Institute:	Yale University
Last Name:	Ye
First Name:	Lupeng
Address:	850 West campus drive
Email:	lupeng.ye@yale.edu
Phone:	2035436568

Subject:

Subject ID:	SU002168
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Genotype	Treatment
SA198305	20190125 CarT_Prodh2_KI_5 M109	PRODH2-CAR	PRODH2
SA198306	20190125 CarT_Prodh2_KI_4 M108	PRODH2-CAR	PRODH2
SA198307	20190125 CarT_Prodh2_KI_1 M105	PRODH2-CAR	PRODH2
SA198308	20190125 CarT_Prodh2_KI_2 M106	PRODH2-CAR	PRODH2
SA198309	20190125 CarT_Prodh2_KI_3 M107	PRODH2-CAR	PRODH2
SA198300	20190125 CarT_Prodh2_KI_Vector1 M105	PRODH2(Stop)-CAR	PRODH2(Stop)
SA198301	20190125 CarT_Prodh2_KI_Vector5 M105	PRODH2(Stop)-CAR	PRODH2(Stop)
SA198302	20190125 CarT_Prodh2_KI_Vector4 M105	PRODH2(Stop)-CAR	PRODH2(Stop)
SA198303	20190125 CarT_Prodh2_KI_Vector2 M105	PRODH2(Stop)-CAR	PRODH2(Stop)
SA198304	20190125 CarT_Prodh2_KI_Vector3 M105	PRODH2(Stop)-CAR	PRODH2(Stop)

Showing results 1 to 10 of 10

Showing results 1 to 10 of 10

Collection:

Collection ID:	CO002161
Collection Summary:	cell were counted, washed twice with PBS then used for metabolite extraction.
Sample Type:	T-cells

Treatment:

Treatment ID:	TR002180
Treatment Summary:	T cells were electroporated with Cas9-RNP, then transduced with CD22-CAR;PRODH2 or PRODH2(Stop) AAV, CAR-T cells were collected for metabolite extraction.

Sample Preparation:

Sampleprep ID:	SP002174
Sampleprep Summary:	After normalizing cell counts, 800 μL of 80 % (vol / vol) HPLC-grade methanol (Sigma) (precooled to -80 oC on dry ice) was added to fresh cells in a 1.5-mL microcentrifuge tube, then tubes were put on dry ice for 30 minutes. The tubes were then incubated on ice for 20 minutes and centrifuged at 15,000 x g for 15 min at 4 oC to pellet the cell debris. The metabolite-containing supernatant was transferred to a new 1.5-mL microcentrifuge tube on dry ice. Metabolite extraction was repeated with 400 μL of 80 % (vol / vol) HPLC-grade methanol. The cell lysate / methanol mixtures were dried by Speedvac at room temperature. The metabolites were dissolved again with 80 % (vol / vol) methanol, then centrifuged at 18,000 x g for 10 min to remove any particulates, and the metabolite mixtures were stored at -80 oC until LC-MS analysis.

Sampleprep ID:

SP002174

Sampleprep Summary:

After normalizing cell counts, 800 μL of 80 % (vol / vol) HPLC-grade methanol (Sigma) (precooled to -80 oC on dry ice) was added to fresh cells in a 1.5-mL microcentrifuge tube, then tubes were put on dry ice for 30 minutes. The tubes were then incubated on ice for 20 minutes and centrifuged at 15,000 x g for 15 min at 4 oC to pellet the cell debris. The metabolite-containing supernatant was transferred to a new 1.5-mL microcentrifuge tube on dry ice. Metabolite extraction was repeated with 400 μL of 80 % (vol / vol) HPLC-grade methanol. The cell lysate / methanol mixtures were dried by Speedvac at room temperature. The metabolites were dissolved again with 80 % (vol / vol) methanol, then centrifuged at 18,000 x g for 10 min to remove any particulates, and the metabolite mixtures were stored at -80 oC until LC-MS analysis.

Combined analysis:

Analysis ID	AN003401
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Agilent 6490
Column	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Agilent 6490 QQQ
Ion Mode	POSITIVE
Units	ppm

Chromatography:

Chromatography ID:	CH002514
Instrument Name:	Agilent 6490
Column Name:	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Chromatography Type:	HILIC

MS:

MS ID:	MS003168
Analysis ID:	AN003401
Instrument Name:	Agilent 6490 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Two metabolomics strategies were adopted, i.e. untargeted metabolomics (aiming to unbiasedly detect all detectable metabolites) and targeted approaches (aiming to detect specifically defined metabolites, such as related metabolites in the proline metabolism and T cell metabolism). For untargeted metabolomics analysis, the optimized workflow consists of automated peak detection and integration, peak alignment, background noise subtraction, and multivariate data analysis. These steps were carried out for comprehensive metabolite phenotyping of the two groups using Agilent Mass Hunter Qualitative Analysis Software (Version B.07.0.0, build 7.0.7024.0) and Agilent Mass Profiler Professional (Version 14.5-Build 2772). The metabolites were first putatively identified based on accurate mass match (accurate mass ± 30 ppm error) and fragmentation pattern match. Putative structural annotation was carried out by searching the metabolite databases HMDB (http://www.hmdb.ca/) and METLIN (http:// metlin.scripps.edu) using the mass-to-charge ratio of the metabolic features.
Ion Mode:	POSITIVE