Differential protein-protein interactions underlie signaling mediated by the TCR and a 4-1BB domain–containing CAR.

Cells rely on activity-dependent protein-protein interactions to convey biological signals. For chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to stimulate the formation of protein complexes similar to those stimulated by T cell receptor (TCR)–mediated signaling, but the number and type of protein interaction–mediating binding domains differ between CARs and TCRs. Here, we performed coimmunoprecipitation mass spectrometry analysis of a second-generation, CD19-directed 4-1BB:ζ CAR (referred to as bbζCAR) and identified 128 proteins that increased their coassociation after target engagement. We compared activity-induced TCR and CAR signalosomes by quantitative multiplex coimmunoprecipitation and showed that bbζCAR engagement led to the activation of two modules of protein interactions, one similar to TCR signaling that was more weakly engaged by bbζCAR as compared with the TCR and one composed of TRAF signaling complexes that was not engaged by the TCR. Batch-to-batch and interindividual variations in production of the cytokine IL-2 correlated with differences in the magnitude of protein network activation. Future CAR T cell manufacturing protocols could measure, and eventually control, biological variation by monitoring these signalosome activation markers. Editor's summary: Chimeric antigen receptor (CAR) T cells are effective therapies against some cancers; however, a better understanding of their signaling pathways would help in enhancing their efficacy. Ritmeester-Loy et al. identified and quantified the protein-protein interactions induced by CAR stimulation in CAR T cells generated from healthy donors. As well as characterizing protein-protein interactions that were induced both by CARs and endogenous T cell receptors, the authors identified a set of protein-protein interactions specific to CARs. The extent of activation of CAR T cells varied from batch to batch but correlated with signaling strength, suggesting that variations in key signaling components may modulate CAR T cell performance. —John F. Foley [ABSTRACT FROM AUTHOR]