No abstract

During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.

Introduction:In order 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 surfacebound T cell receptors (TCRs), binding to peptide-major histocompatibility complexes (pMHC). With some temporal latency, this binding event induces TCR signaling and T cell effector function. In order for TCR recognition to take place, T cells must efficiently survey surfaces of antigen presenting cells (APCs), which may display mainly non-stimulatory pMHC 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 tradeoff 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 3-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 pMHC, but their strategy has not been amenable to study. We used time-resolved lattice light sheet (LLS) microscopy and Qdot-enabled synaptic contact mapping (SCM) microscopy to show how microvilli on the surface of T cells search opposing cells and surfaces prior to 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 one minute, which is equivalent to the roughly one 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 non-synapse 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 pMHC and increased persistence of TCR-occupied contacts. Microvillar scanning in synapse regions lacking pMHC showed dynamics similar to non-synaptic 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 ac...

Highlights d Myeloid cells retain tumor-derived antigens in intracellular vesicles d Dynamic synapses facilitate membrane and vesicular exchange between myeloid cells d Migratory DC pass vesicular tumor antigen to resident DC at points of contact d Migratory versus resident cDC1 exhibit differential T cell priming characteristics

Interactions among drugs play a critical role in the killing efficacy of multidrug treatments. Recent advances in theory and experiment for three-drug interactions enable the search for emergent interactions-ones not predictable from pairwise interactions. Previous work has shown it is easier to detect synergies and antagonisms among pairwise interactions when a rescaling method is applied to the interaction metric. However, no study has carefully examined whether new types of normalization might be needed for emergence. Here, we propose several rescaling methods for enhancing the classification of the higher order drug interactions based on our conceptual framework. To choose the rescaling that best separates synergism, antagonism and additivity, we conducted bacterial growth experiments in the presence of single, pairwise and triple-drug combinations among 14 antibiotics. We found one of our rescaling methods is far better at distinguishing synergistic and antagonistic emergent interactions than any of the other methods. Using our new method, we find around 50% of emergent interactions are additive, much less than previous reports of greater than 90% additivity. We conclude that higher order emergent interactions are much more common than previously believed, and we argue these findings for drugs suggest that appropriate rescaling is crucial to infer higher order interactions.