Visualizing dynamic microvillar search and stabilization during ligand detection by T cells.

INTRODUCTION For T cells to mount an adaptive immune response and enact cell-mediated immunity, they must first successfully detect rare cognate antigen. This detection is achieved by surface-bound T cell receptors (TCRs), binding to peptide-loaded major histocompatibility complexes (pMHCs). With some temporal latency, this binding event induces TCR signaling and T cell effector function. For TCR recognition to take place, T cells must efficiently survey surfaces of antigen-presenting cells (APCs), which may display mainly nonstimulatory pMHCs and only rare cognate antigen in a process involving close (nanometer-scale) membrane apposition. Additionally, those rare pMHC ligands are distributed nonuniformly on subsets of APCs and only within specific lymph nodes. Thus, T cells must solve a classic search trade-off between speed and sensitivity: Faster movements provide larger overall coverage with costs at the level of sensitivity. Successful search, which results in ligand detection, is ultimately required for effector function and T cell–mediated adaptive immune response. Although surface deformations are indicated in this recognition process, the full understanding of search strategy requires real-time full three-dimensional analysis that has not been possible using fixed or low-resolution approaches. RATIONALE It has long been supposed that small microvilli on T cell surfaces are used as sensory organs to enable the search for pMHCs, but their strategy has not been amenable to study. We used time-resolved lattice light-sheet (LLS) microscopy and quantum dot–enabled synaptic contact mapping (SCM) microscopy to show how microvilli on the surface of T cells search opposing cells and surfaces before and during antigen recognition. RESULTS In characterizing microvilli movement on T cell surfaces, we uncovered fractal organization of the microvilli, suggesting consistent coverage across scales. We found that their movements surveyed the majority of opposing space within 1 minute, which is equivalent to the roughly 1-minute half-life of T cell–APC contacts in vivo. Individual microvilli local dwell times were sufficiently long to permit discrimination of pMHC half-lives. Protrusion density was similar in nonsynapse and synapse regions and did not change appreciably during synapse development, suggesting that T cells did not “intensify” search upon recognition. TCR recognition resulted in selective stabilization of receptor-occupied protrusions as seen by longer microvilli dwell times in synapse regions with cognate pMHCs and increased persistence of TCR-occupied contacts. Microvillar scanning in synapse regions lacking cognate pMHCs showed dynamics similar to nonsynaptic regions, supporting the dependence of TCR stabilization on ligand recognition. Subsequent TCR movements took place upon the stabilized protrusions, even while transient ones tested new regions. In the absence of tyrosine kinase signaling, microvillar search and TCR-occupied protrusion stabilization continued. Intrinsic stabilization was also independent of the actin cytoskeleton, suggesting that the process selects for dense avid TCR microclusters. CONCLUSION Intercellular receptor complex formation takes place on a rapidly evolving three-dimensional surface under time constraints imposed by a cell’s dynamic movements. This work defines the efficient cellular search process against which ligand detection takes place in T cells. Microvillar movements were capable of nearly complete scanning of APCs at physiological T-APC contact durations while maintaining microvilli dwell times long enough to differentiate short-lived antagonist interactions from longer-lived agonist interactions. Stabilization of microvilli required the presence of both TCR and cognate pMHCs but was independent of downstream tyrosine kinase signaling and the actin cytoskeleton. Based on these findings, the palpation of opposing cell surfaces by dynamic microvilli on T cells underlies TCR recognition. These microvillar dynamics impose a time pressure for ligands to solidify interactions with an opposing surface. This work lays down the framework for topological scan in T cell–APC recognition. Additionally, an understanding of the role that active surface topology plays in ligand detection could also shed light on cell-cell recognition in other physiological systems.