{"doi":"10.1101/2025.07.04.663030","title":"Inclusion of the ζ-chain drives phosphotyrosine signalling in CD19-CAR T cells","abstract":"Although chimeric antigen receptor (CAR) T cell therapy has revolutionised individualised cancer therapies for relapsed/refractory lymphomas, signalling mechanisms underlying CAR T activation remain incompletely understood, especially among the three generations of CAR T exploiting different signalling domains. Here, using Jurkat T cell as a model, we investigate how costimulation influences 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 find that including the ζ-chain in first (ζ-CAR), second (28ζ-CAR and BBζ-CAR), and third (28BBζ-CAR) generation CARs largely determines pY signalling, irrespective of costimulation. Further, we show that the phosphatase activity of PTPN22 and SHP-1 are largely negligible for activation of CARs, but indiscriminate inhibition of phosphatases using Pervanadate (PV) selectively activates BBζ-CARs without antigen encounter. Finally, we find that selective, partial inhibition of Itk using Soquelitinib reduces basal CD69 expression in Jurkat CAR T cells while maintaining their ability to activate in response to antigen. Our data suggest that the ζ-chain determines the pY signalling profile of CD19-CAR Jurkat T cells and that Itk may drive antigen-independent CD19-CAR activation.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":558438,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9513,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":363861,"name":"Ryan Z. Puterbaugh","orcid":"0000-0002-6412-8703","position":1,"is_corresponding":false},{"id":1441858,"name":"Tae Suk Ro","orcid":null,"position":2,"is_corresponding":false},{"id":624028,"name":"Xinyan Zhang","orcid":"0000-0001-6269-3138","position":3,"is_corresponding":false},{"id":301480,"name":"Xiaolei Su","orcid":"0000-0001-8696-7922","position":4,"is_corresponding":false},{"id":347401,"name":"Arthur R. Salomon","orcid":"0000-0002-7223-8996","position":5,"is_corresponding":false},{"id":822196,"name":"Kenneth P. Callahan","orcid":"0000-0003-4848-1512","position":0,"is_corresponding":true}],"reference_count":55,"raw_metadata":null,"created_at":"2026-07-19T02:55:25.969263Z","pmid":"40672291","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}