Phosphoproteomic analysis of successive Jurkat CD19-CAR generations reveals TCRζ-driven signalling.

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.