Figure 2. An Integrated Mechanistic Model of CAR Signaling Initiation.

Research on T-cell receptor (TCR) triggering and the specific signaling domains utilized in CARs suggests the following potential mechanisms working in concert to initiate CAR signaling. (A) Ligand binding could generate mechanical forces that lead to the dissociation of CAR intracellular domains from the plasma membrane, thereby unmasking critical binding sites for downstream signaling molecules. (i) At rest, CAR intracellular domains (e.g. CD28 and CD3ζ) may interact with the plasma membrane, as they do in their native receptor contexts, through basic residue motifs that bind to the negatively charged inner leaflet of the plasma membrane [14–16]. (ii) Upon antigen binding, CAR intracellular domains dissociate from the plasma membrane and adopt a signaling-competent conformation that allows interactions with downstream signaling molecules, including kinases such as ZAP-70 and Lck [15,16]. Phosphorylation of the intracellular domains is thought to lock the domains in the membrane-free state [15]. (B) Extending the receptor deformation model of TCR triggering to CARs suggests that the changes in CAR conformation from (A, i) to (A, ii) may arise from mechanical pulling or pushing between the T cell and the target cell. (i) A pulling force can be transmitted via tension in the CAR extracellular and transmembrane domains to dislodge the intracellular domains from the plasma membrane. (ii) A pushing force may alter the local membrane curvature, thereby reducing the stability of the membrane-associated state of the CAR intracellular domains. (C) In the kinetic segregation model of TCR triggering, bulky phosphatases must be physically segregated from TCRs for T-cell activation domains to transduce signal. Thus, in addition to having accessible (i.e. membrane-free) intracellular signaling domains, CARs may also need to be segregated from phosphatases to initiate signal transduction. (i) Segregation of phosphatases and CARs can occur when CAR/ligand interactions force the T cell and target cell into close apposition and exclude bulky phosphatases from the immunological synapse (IS). (ii) By this logic, CARs with excessively long extracellular spacers that allow phosphatases to comingle with CARs at the IS would not be able to robustly activate T-cell signaling. (D) CARs in resting T cells are localized diffusely together with other surface receptors such as CD45. As the events in (C) take place in response to target-cell engagement, ligated CARs can coalesce into microclusters, which have been confirmed to exclude CD45 and transduce T-cell activation signals [26]. With time, microclusters at the CAR-containing immunological synapse are hypothesized to coalesce and organize with other native surface receptors into the supramolecular activation cluster (SMAC), commonly observed at TCR synapses.