Visualizing Dynamic Microvillar Search and Stabilization during Ligand Detection by T Cells

Abstract

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...

Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering

“…Inter-NP spacing from 40 nm to 120 nm, cluster sizes from 240 nm to 1440 nm diameter, and densities from ~100/µm 2 to ~30/µm 2 were compared (Fig 2a). The pY signal on all patterns was multifocal, likely reflecting natural patterns of small, F-actin-dependent projections that mediate first contact of T cells with the surface 26 . The pY signal was strongly colocalized with the UCHT1 Fab’ clusters regardless of the geometry (Fig.…”

Molecular mechanisms of T cell sensitivity to antigen

“…Instead of waiting for passive close contact formation and hence TCR/pMHC binding, T cells actively produce close contacts. They use structures known as microvilli or invadosome‐like protrusions (ILPs) . These actin‐rich 100‐200 nm diameter protrusions enable T cells to push on nearby cells in order to achieve close membrane apposition compatible with TCR/pMHC interactions, which leads to CD45 segregation (Figure B).…”

“…They use structures known as microvilli or invadosome‐like protrusions (ILPs) . These actin‐rich 100‐200 nm diameter protrusions enable T cells to push on nearby cells in order to achieve close membrane apposition compatible with TCR/pMHC interactions, which leads to CD45 segregation (Figure B). It is noteworthy that these structures have short (approximately 5‐50 seconds) lifetimes without antigen, but upon antigen recognition, they can be stabilized for long (approximately 10 minutes) timescales .…”

“…These actin‐rich 100‐200 nm diameter protrusions enable T cells to push on nearby cells in order to achieve close membrane apposition compatible with TCR/pMHC interactions, which leads to CD45 segregation (Figure B). It is noteworthy that these structures have short (approximately 5‐50 seconds) lifetimes without antigen, but upon antigen recognition, they can be stabilized for long (approximately 10 minutes) timescales . This stabilization may involve TCR signaling leading to sustained actin polymerization, a phenomenon observed in these protrusions …”

Mechanosensing in the immune response

“…The role of cytoskeletal forces for kinetic segregation, along with associated kinetic proofreading models for TCR signaling have been exhaustively reviewed in Comrie et al [88]. Further evidence for active formation of microclusters comes from a recent study which used high-resolution light sheet microscopy to show that dynamic microvilli on T cells drive the formation of close contacts on the APC surface, which are colocalized with TCR microclusters [89]. This is consistent with earlier work showing the correlation between microclusters and regions of reduced membrane fluctuations [84].…”