PXD007921

PXD007921 is an original dataset announced via ProteomeXchange.

Title	Global phosphoproteomic analysis of signaling by distinct chimeric antigen receptors reveals kinetic and quantitative differences that influence cell function
Description	Chimeric antigen receptors (CARs) are synthetic proteins that redirect T cell specificity by linking an extracellular ligand binding domain to intracellular T cell signaling domains. CAR-expressing T (CAR-T) cells have demonstrated significant efficacy for the treatment of refractory B cell malignancies and are being evaluated as immunotherapeutic reagents for many other cancers. CAR designs are based on the fundamental principles of TCR recognition and most CARs employ the T cell-activating CD3z endodomain alongside a costimulatory domain from CD28 or 4-1BB. However, emerging data suggest that CD28/CD3z and 4-1BB/CD3z signaling modules promote divergent metabolic pathways, gene expression programs, and cell fates. To determine how CAR phosphoprotein signaling drives these disparate cell fates, we analyzed CAR ligation-induced signaling networks in primary human T cells using shotgun mass spectrometry. We isolated CD8+CD62L+ T cells from healthy donors and introduced a CD28/CD3z or 4-1BB/CD3z CAR by lentiviral transduction. Transduced T cells were purified by FACS and expanded once in vitro. When the cells returned to a resting state, CD28/CD3z or 4-1BB/CD3z CAR-T cells were stimulated for 10 or 45 minutes with magnetic microbeads coated with a monoclonal antibody specific for a 9 amino acid tag in the CAR extracellular sequence. CAR-T cells were also left unstimulated for 10 or 45 minutes to serve as controls. Altogether, 8 unique conditions were tested in an experiment and three independent experiments were performed.
HostingRepository	PRIDE
AnnounceDate	2018-08-13
AnnouncementXML	Submission_2018-08-13_10:24:50.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Jacob Kennedy
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	carbamoylated residue; phosphorylated residue
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2017-10-09 02:39:09	ID requested
⏵ 1	2018-08-13 10:24:51	announced

Dataset with its publication pending

curator keyword: Biological, Biomedical

submitter keyword: Chimeric antigen receptor, T cell, primary T cell, phosphoprotein signaling, basic reverse phase fractionation, IMAC enrichment, phosphoproteomics, LC-MS/MS

Stanley Riddell
contact affiliation	Immunology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA
contact email	sriddell@fredhutch.org
lab head
Jacob Kennedy
contact affiliation	Fred Hutchinson Cancer Research Center
contact email	jkennedy@fhcrc.org
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD007921
     1. Label: PRIDE project
     2. Name: Global phosphoproteomic analysis of signaling by distinct chimeric antigen receptors reveals kinetic and quantitative differences that influence cell function