Dynamic microvillar search in traditional and synthetic ligand detection by T cells

A T cell’s ability to efficiently surveil its environment and recognize and respond appropriately to cognate antigen via its antigen receptor is a critical feature of adaptive immunity. Although cell-cell interactions are often depicted as two flat surfaces pushing up against each other with molecules moving laterally within a single plane, the reality is that T cells must probe three-dimensionally complex surfaces covered in relatively large, glycosylated molecules as they migrate through lymph nodes and other tissues during immune surveillance. Yet, it is unknown how T cells overcome these barriers to make very close contact with apposing membranes, bringing together the small membrane receptors that initiate T cell signaling. Prior to these studies, whether T cells use their membrane microvilli to aid in detection of antigen was largely unknown. Here, we used lattice light sheet (LLS) microscopy and synaptic contact mapping (SCM) total internal reflection fluorescence (TIRF) imaging to visualize microvilli on T cells with high spatiotemporal resolution prior to and during ligand detection by T cells. We found that T cell microvilli are highly dynamic structures that efficiently probe surfaces in physiologically relevant timescales. Specifically upon binding of antigen receptor to cognate ligand, the underlying microvillus became stabilized, providing a persistent surface for signaling. Of note, ZAP70 signaling and actin polymerization were not required to maintain these close contacts after binding to cognate antigen, indicating that physical pinning of membranes via membrane receptors is a significant contributor to changes in membrane dynamics. Given that natural antigen receptors make use of this cell biology, we then asked whether engineered chimeric antigen receptors (CARs) interact similarly. We found that CARs distributed in the plasma membrane similarly to natural T cell receptors (TCRs) although notably in distinct patches. However, when engaging ligands these induced hyper-stabilization of the underlying microvilli relative to that of the TCR. This hyper-stabilization was dependent on the high affinity and avidity of CAR binding and was associated with altered organization of molecules at the cell-cell interface, decreased effector function, and increased propensity for exhaustion. Thus, although microvillar search is involved in both natural and synthetic ligand detection, microvillar dynamics are differentially altered depending on strength of receptor binding. This work reveals the cell biology underlying T cell ligand detection and its sensitivity to changes in binding dynamics, with likely clinical importance for the development of cell therapies