PXD065669

PXD065669 is an original dataset announced via ProteomeXchange.

Title	Phosphoproteomic analysis of successive Jurkat CD19-CAR generations reveals TCRζ-driven signalling
Description	Although chimeric antigen receptor (CAR) T cell therapy has revolutionised individualised cancer therapies for relapsed/refractory lymphomas, low long-term retention due to basal signalling (antigen-independent activation in the absence of cognate antigen) and off-target toxicity limit the broad applicability of CAR-T products. During CAR development, researchers use model systems, like Jurkat T cells (Jurkats), to screen intracellular signalling arrangements based on their ability to activate (e.g., CD69 expression) and withstand repeated antigen encounters. Although Jurkats are standard for CAR screening, the mapping of CAR generations to Jurkat-specific pTyr networks relative to key TCR nodes and CD69 readouts is not well defined, blurring how hierarchical signalling drives activation. Here, we investigated how costimulation influenced tyrosine phosphorylation cascades using LC-MS/MS based phosphotyrosine (pY) proteomics and CD69 expression in the presence of small molecule inhibitors of key TCR signalling regulators. We found that including TCRζ (CD3ζ; gene CD247) in first (ζ-CAR), second (28ζ-CAR and BBζ-CAR), and third (28BBζ-CAR) generation CARs largely determined pY signalling, irrespective of costimulation. Further, we showed that the phosphatase activity of PTPN22 and SHP-1 were largely negligible for activation of CARs, but indiscriminate inhibition of phosphatases using pervanadate (PV) selectively activated BBζ-CARs without antigen encounter. Finally, we found that selective, partial inhibition of Itk using Soquelitinib reduced basal CD69 expression in CAR-Jurkat cells while maintaining their ability to activate in response to antigen. These data suggest that TCRζ determines the pY signalling profile and that Itk drives basal activation of CD19-CAR Jurkats, which may impact evaluation of new CAR designs in CAR-Jurkat screens.
HostingRepository	PRIDE
AnnounceDate	2025-11-21
AnnouncementXML	Submission_2025-11-21_11:16:26.931.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Arthur Salomon
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	phosphorylated residue
Instrument	Orbitrap Ascend

Revision	Datetime	Status	ChangeLog Entry
0	2025-07-01 19:13:17	ID requested
⏵ 1	2025-11-21 11:16:27	announced

Callahan A, Puterbaugh RZ, Ro T, Zhang X, Su X, Salomon AR, -driven signalling. Cell Signal, 138():112204(2026) [pubmed]

10.1016/j.cellsig.2025.112204;

submitter keyword: None

Arthur Salomon
contact affiliation	Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, 02903
contact email	art@drsalomon.com
lab head
Arthur Salomon
contact affiliation	Brown University
contact email	art@drsalomon.com
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/11/PXD065669

PRIDE project URI

• PRIDE
  1. PXD065669
     1. Label: PRIDE project
     2. Name: Phosphoproteomic analysis of successive Jurkat CD19-CAR generations reveals TCRζ-driven signalling