PT  - JOURNAL ARTICLE
AU  - Samuel A. Ritmeester-Loy
AU  - Jonathan D Lautz
AU  - Yue Zhang-Wong
AU  - Joshua Gustafson
AU  - Ashley Wilson
AU  - Chenwei Lin
AU  - Philip R. Gafken
AU  - Michael C Jensen
AU  - Rimas Orentas
AU  - Stephen E.P. Smith
TI  - State-dependent protein-protein interactions mediating 4-1BB CAR Signaling
AID  - 10.1101/2022.06.07.495180
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.06.07.495180
4099  - http://biorxiv.org/content/early/2022/06/09/2022.06.07.495180.short
4100  - http://biorxiv.org/content/early/2022/06/09/2022.06.07.495180.full
AB  - Cells rely on activity-dependent protein-protein interactions to convey biological signals, but the state-dependent interactome is notoriously cell-specific and undercharacterized1. In the case of chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to trigger the formation of protein complexes similar to those triggered 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 co-immunoprecipitation mass spectrometry of a 2nd generation CD19-directed 4-1BB:zeta CAR (referred to as bbζCAR) and identified 67 proteins that increased their co-association after target engagement. We compared activity-induced TCR and CAR signalosomes using quantitative multiplex co-immunoprecipitation and showed that bbζCAR engagement leads to activation of two modules of protein interactions, one similar to TCR signaling that is more weakly engaged in bbζCAR vs. TCR, and one composed of TRAF signaling complexes that is not engaged by the TCR. Batch-to-batch and inter-individual variations in IL2 production 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.One Sentence Summary We define a network of protein interactions engaged by chimeric antigen receptors following target binding, and show that the magnitude of network activation correlates with IL-2 secretion, a proxy measure for CAR T cell function.Competing Interest StatementThe authors have declared no competing interest.