Audrey Gérard

The Par polarity complex regulates Rap1- and chemokine-induced T cell polarization

Cell polarization is required for virtually all functions of T cells, including transendothelial migration in response to chemokines. However, the molecular pathways that establish T cell polarity are poorly understood. We show that the activation of the partitioning defective (Par) polarity complex is a key event during Rap1- and chemokine-induced T cell polarization. Intracellular localization and activation of the Par complex are initiated by Rap1 and require Cdc42 activity. The Rac activator Tiam1 associates with both Rap1 and components of the Par complex, and thereby may function to connect the Par polarity complex to Rap1 and to regulate the Rac-mediated actin remodelling required for T cell polarization. Consistent with these findings, Tiam1-deficient T cells are impaired in Rap1- and chemokine-induced polarization and chemotaxis. Our studies implicate Tiam1 and the Par polarity complex in polarization of T cells, and provide a mechanism by which chemokines and Rap1 regulate T cell polarization and chemotaxis.

Visualization of immediate immune responses to pioneer metastatic cells in the lung

Lung metastasis is the lethal determinant in many cancers and a number of lines of evidence point to monocytes and macrophages having key roles in its development. Yet little is known about the immediate fate of incoming tumour cells as they colonize this tissue, and even less known about how they make first contact with the immune system. Primary tumours liberate circulating tumour cells (CTCs) into the blood and we have developed a stable intravital two-photon lung imaging model in mice for direct observation of the arrival of CTCs and subsequent host interaction. Here we show dynamic generation of tumour microparticles in shear flow in the capillaries within minutes of CTC entry. Rather than dispersing under flow, many of these microparticles remain attached to the lung vasculature or independently migrate along the inner walls of vessels. Using fluorescent lineage reporters and flow cytometry, we observed ‘waves’ of distinct myeloid cell subsets that load differentially and sequentially with this CTC-derived material. Many of these tumour-ingesting myeloid cells collectively accumulated in the lung interstitium along with the successful metastatic cells and, as previously understood, promote the development of successful metastases from surviving tumour cells. Although the numbers of these cells rise globally in the lung with metastatic exposure and ingesting myeloid cells undergo phenotypic changes associated with microparticle ingestion, a consistently sparse population of resident conventional dendritic cells, among the last cells to interact with CTCs, confer anti-metastatic protection. This work reveals that CTC fragmentation generates immune-interacting intermediates, and defines a competitive relationship between phagocyte populations for tumour loading during metastatic cell seeding.

The Rac activator Tiam1 controls efficient T-cell trafficking and route of transendothelial migration

Migration toward chemoattractants is a hallmark of T-cell trafficking and is essential to produce an efficient immune response. Here, we have analyzed the function of the Rac activator Tiam1 in the control of T-cell trafficking and transendothelial migration. We found that Tiam1 is required for chemokine- and S1P-induced Rac activation and subsequent cell migration. As a result, Tiam1-deficient T cells show reduced chemotaxis in vitro, and impaired homing, egress, and contact hypersensitivity in vivo. Analysis of the T-cell transendothelial migration cascade revealed that PKCzeta/Tiam1/Rac signaling is dispensable for T-cell arrest but is essential for the stabilization of polarization and efficient crawling of T cells on endothelial cells. T cells that lack Tiam1 predominantly transmigrate through individual endothelial cells (transcellular migration) rather than at endothelial junctions (paracellular migration), suggesting that T cells are able to change their route of transendothelial migration according to their polarization status and crawling capacity.

Regulation of T cell priming by lymphoid stroma.

The priming of immune T cells by their interaction with dendritic cells (DCs) in lymph nodes (LN), one of the early events in productive adaptive immune responses, occurs on a scaffold of lymphoid stromal cells, which have largely been seen as support cells or sources of chemokines and homeostatic growth factors. Here we show that murine fibroblastic reticular cells (FRCs), isolated from LN of B6 mice, play a more direct role in the immune response by sensing and modulating T cell activation through their upregulation of inducible nitric oxide synthase (iNOS) in response to early T cell IFNγ production. Stromal iNOS, which only functions in very close proximity, attenuates responses to inflammatory DC immunization but not to other priming regimens and preferentially affects Th1 cells rather than Th2. The resultant nitric oxide production does not affect T cell-DC coupling or initial calcium signaling, but restricts homotypic T cell clustering, cell cycle progression, and proliferation. Stromal feedback inhibition thus provides basal attenuation of T cell responses, particularly those characterized by strong local inflammatory cues.

Secondary T cell-T cell synaptic interactions drive the differentiation of protective CD8 + T cells

Immunization results in the differentiation of CD8+ T cells, such that they acquire effector abilities and convert into a memory pool. Priming of T cells takes place via an immunological synapse formed with an antigen-presenting cell (APC). By disrupting synaptic stability at different times, we found that the differentiation of CD8+ T cells required cell interactions beyond those made with APCs. We identified a critical differentiation period that required interactions between primed T cells. We found that T cell–T cell synapses had a major role in the generation of protective CD8+ T cell memory. T cell–T cell synapses allowed T cells to polarize critical secretion of interferon-γ (IFN-γ) toward each other. Collective activation and homotypic clustering drove cytokine sharing and acted as regulatory stimuli for T cell differentiation.

Cutting edge: Dok-1 and Dok-2 adaptor molecules are regulated by phosphatidylinositol 5-phosphate production in T cells.

Downstream of tyrosine kinase (Dok) proteins Dok-1 and Dok-2 are involved in T cell homeostasis maintenance. Dok protein tyrosine phosphorylation plays a key role in establishing negative feedback loops of T cell signaling. These structurally related adapter molecules contain a pleckstrin homology (PH) domain generally acting as a lipid/protein-interacting module. We show that the presence of this PH domain is necessary for the tyrosine phosphorylation of Dok proteins and their negative functions in T cells. We find that Dok-1/Dok-2 PH domains bind in vitro to the rare phosphoinositide species, phosphatidylinositol 5-phosphate (PtdIns5P). Dok tyrosine phosphorylation correlates with PtdIns5P production in T cells upon TCR triggering. Furthermore, we demonstrate that PtdIns5P increase regulates Dok tyrosine phosphorylation in vivo. Together, our data identify a novel lipid mediator in T cell signaling and suggest that PH-PtdIns5P interactions regulate T cell responses.

Functional interaction of RasGAP-binding proteins Dok-1 and Dok-2 with the Tec protein tyrosine kinase

The Dok adaptor family of proteins binding to RasGAP, consisting of Dok-1 and Dok-2, are critical regulators in cell proliferation. These molecules are partners and/or substrates of different protein tyrosine kinases considered as oncoproteins. Here, we show that Dok-1 and Dok-2 are the major tyrosine-phosphorylated proteins associated to Tec, a protein tyrosine kinase expressed in T cells. Furthermore, we evaluate the effect of Dok-1 or Dok-2 on Tec-mediated signalling pathways in T cells. Here, we provide evidence that Dok-1 and Dok-2 proteins are involved in a negative feedback regulation of Tec via a downregulation of its tyrosine phosphorylation and downstream signalling pathways including the Ras pathway. Either Dok-1 or Dok-2 therefore represents a mean of potent retrograde control for protein tyrosine kinase signalling, and then possibly of tumor development.

DOK4 and DOK5: new Dok-related genes expressed in human T cells.

Dok proteins are adapter proteins involved in signal transduction. Several intracellular proteins expressed in lymphocytes meet the criteria of membrane-associated adapter proteins such as members of the Dok family. To understand the role and the formation of multiprotein networks involving Dok proteins in T lymphocytes, we search for potential additional members of this family. Here, we describe the two new human dok-related genes DOK4 and DOK5 and present data showing the expression of DOK4 and DOK5 genes in T cells. These genes are the orthologues of mouse Dok4 and Dok5 genes. Based on analysis of phylogenetic trees and exon/intron structure of Dok family members, DOK4 and DOK5 define a subfamily within dok genes distinct from DOK1, DOK2 and DOK3. So, Dok-4 and Dok-5 molecules constitute a new group of adapter proteins in T cells, requiring further functional analysis.

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

Search and capture in space and time How immunological T cells scan target cells for ligands is poorly understood. Cai et al. examined microvillar dynamics in living T cells in three dimensions and real time. The T cells palpated all spots on a surface within about 1 min through rapid movements of their microvilli. The time it took to scan the surface matched the movement rate of cells through tissues. These contacts took place in the absence of T cell receptor recognition and were stabilized independently of signaling or the cytoskeleton. Instead, stabilization depended on ligand affinity. The findings explain why many of the previously described components of the immunological synapse and T cell receptor signaling reside on three-dimensional microvillar-derived projections. Science, this issue p. eaal3118 Immunological T cells palpate opposing cell surfaces in their search for ligands. 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. Dynamics of T cell antigen search. (A) Microvilli distribute with fractal organization on T cell surfaces and efficiently scan their surroundings. Microvilli are stabilized in the immunological synapse upon antigen recognition. (B) Overlays of an isolated T cell at three time points indicate the dynamic nature of microvilli

The SH3 domain of Tec kinase is essential for its targeting to activated CD28 costimulatory molecule

The Tec family of protein tyrosine kinases plays an important role in T cell signaling. Tec, the prototypical member of this kinase family, can interact with CD28, which is a costimulatory molecule. However, the regulation of Tec upon CD28 stimulation remains poorly understood. Here we show that CD28–B7‐mediated interactions are likely involved in the relocalization of Tec at the contactzone between T cells and APC. Upon CD28 ligation with specific antibodies or natural ligands, Tec translocates to the plasma membrane where it colocalizes with the CD28 molecule. The Src‐homology 3(SH3) domain of Tec and the two proline‐rich motifs of CD28 are involved in this process. Furthermore, we show that CD28 signaling requires the SH3 domain of Tec as well as proline residues presentin the intracytoplasmic tail of CD28. These results should provide new insights into the complex regulation of Tec kinases in T cells.