The CAR Immune Synapse Interactome: How Membrane Organization Shapes CAR T–Tumor Cell Interactions

Unlike conventional drugs, whose activity is shaped by binding to a single target, CAR T-cell (CART) engagement with tumor cells involves multiple receptors, adhesion molecules, and signaling proteins. These components are partially organized within cholesterol-rich membrane microdomains called lipid rafts, forming the CAR immune synapse (CARIS)—the interface through which a CART recognizes, attaches to, and kills a tumor cell.

To evaluate CARIS as a pharmacological interface, we compared CD28- and 4-1BB-CARTs, which exhibit distinct therapeutic profiles. The rapid antitumor activity of CD28-CARTs was associated with brief, dynamic synapses supporting rapid signaling and serial killing. Conversely, 4-1BB-CARTs formed longer, mechanically stable synapses consistent with their slower but sustained activity. Synapse strength was measured using z-Movi acoustic force microscopy.

Lipid rafts were enriched at CARIS. Attenuating membrane lipid raft cholesterol preferentially compromised 4-1BB CARIS mechanical stability, while predominantly impairing CD28 CARIS activation and cytolytic output. The molecular networks underlying these differences were mapped by integrating lipid-raft lipidomics, differential SILAC proteomics to distinguish CART-derived from tumor-derived components, and T-cell transcriptomics.

CD28-associated lipid rafts were enriched in diacylglycerol signaling components, PKCθ, small GTPases, Src-family kinases, and AKT/PI3K-proximal modules, supporting rapid signal-to-function coupling. Stomatin-family and flotillin scaffolds favored dynamic remodeling, while CD8-dominant transcriptional programs supported cytolytic degranulation. Engaged tumor cells displayed membrane-stress signatures consistent with late-stage senescence and apoptotic commitment.

In contrast, 4-1BB-associated lipid rafts were enriched in lysophosphatidic acid, an inhibitory lipid known to suppress CD8 effector functions while promoting integrin-dependent adhesion, consistent with the marked enrichment of integrins at CARIS, NF-κB and cytokine signaling components, and ERM scaffolds that couple lipid rafts to cortical actin, sustaining CARIS stability. Engaged tumor cells exhibited signatures of early apoptotic resistance, including damage-response and repair markers.

Together, these findings establish CARIS as a central determinant of CART function and therapeutic behavior. Mapping and manipulating its lipid–protein networks provide a platform for understanding CART pharmacodynamics and developing strategies to improve therapeutic efficacy.

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