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T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin‐related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F‐actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3‐knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon‐like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three‐dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3‐dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities., Quantification of cytotoxic effector T lymphocytes (CTLs) morphology using standard biomedical assays and developed image analysis algorithms showed that the reduction of actin‐related protein (Arp)3 levels impaired F‐actin content maintenance, leading to defects in CTL functionality. The knockdown CTL motility mode, instead of the actin‐rich lamellipodia‐based migratory strategy, displays blebbing‐like migration at the cost of reduced migration speed. Thus, the role of the Arp2/3 complex is not redundant among the various mechanochemical coordinators involved in the leading‐edge formation in CTLs.

Cell migration is essential for physiological processes as diverse as development, immune defence and wound healing. It is also a hallmark of cancer malignancy. Thousands of publications have elucidated detailed molecular and biophysical mechanisms of cultured cells migrating on flat, 2D substrates of glass and plastic. However, much less is known about how cells successfully navigate the complex 3D environments of living tissues. In these more complex, native environments, cells use multiple modes of migration, including mesenchymal, amoeboid, lobopodial and collective, and these are governed by the local extracellular microenvironment, specific modalities of Rho GTPase signalling and non-muscle myosin contractility. Migration through 3D environments is challenging because it requires the cell to squeeze through complex or dense extracellular structures. Doing so requires specific cellular adaptations to mechanical features of the extracellular matrix (ECM) or its remodelling. In addition, besides navigating through diverse ECM environments and overcoming extracellular barriers, cells often interact with neighbouring cells and tissues through physical and signalling interactions. Accordingly, cells need to call on an impressively wide diversity of mechanisms to meet these challenges. This Review examines how cells use both classical and novel mechanisms of locomotion as they traverse challenging 3D matrices and cellular environments. It focuses on principles rather than details of migratory mechanisms and draws comparisons between 1D, 2D and 3D migration.

A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host’s immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on mouse dendritic cells (DCs) as a binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of the pathogenic strain CFT073 to CD14 reduced DC migration by overactivation of integrins and blunted expression of co-stimulatory molecules by overactivating the NFAT (nuclear factor of activated T-cells) pathway, both rate-limiting factors of T cell activation. This response was binary at the single-cell level, but averaged in larger populations exposed to both piliated and non-piliated pathogens, presumably via the exchange of immunomodulatory cytokines. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn’s disease.

Lymph nodes (LNs) comprise two main structural elements: Fibroblastic reticular cells (FRCs) that form dedicated niches for immune cell interaction and capsular fibroblasts that build a shell around the organ. While LNs are fairly stable in size during homeostatic conditions, immunological challenge causes more than 10-fold increase in size within only a few days. How a solid organ can accommodate such extreme volumetric changes is poorly understood. Here, we characterize the biomechanics of LN swelling on the cellular and organ scale. We identify lymphocyte trapping by influx and proliferation as drivers of an outward pressure force, causing FRCs and their associated conduits to stretch. After an initial phase of relaxation, FRCs sense the resulting strain via cell matrix adhesions, which coordinates local growth and remodeling of the stromal network. While the expanded FRC network adopts its typical configuration, a massive fibrotic reaction of the organ capsule sets in and counters further organ expansion. Thus, different fibroblast populations mechanically control LN swelling in a multi-tier fashion.

Summary Lymphocyte homeostasis and immune surveillance require that T and B cells continuously recirculate between secondary lymphoid organs. Here, we used intravital microscopy to define lymphocyte trafficking routes within the spleen, an environment of open blood circulation and shear forces unlike other lymphoid organs. Upon release from arterioles into the red pulp sinuses, T cells latched onto perivascular stromal cells in a manner that was independent of the chemokine receptor CCR7 but sensitive to Gi protein-coupled receptor inhibitors. This latching sheltered T cells from blood flow and enabled unidirectional migration to the bridging channels and then to T zones, entry into which required CCR7. Inflammatory responses modified the chemotactic cues along the perivascular homing paths, leading to rapid block of entry. Our findings reveal a role for vascular structures in lymphocyte recirculation through the spleen, indicating the existence of separate entry and exit routes and that of a checkpoint located at the gate to the T zone., Graphical Abstract, Highlights • Perivascular pathways support T cell entry into splenic T zones, but not egress from them • Attachment to the homing paths requires activation of GPCRs other than CCR7 • CCR7 mediates one-directional migration and entry into T zones • Inflammation leads to modification of the homing paths and to rapid block of entry, Lymphocyte recirculation between secondary lymphoid organs is critical for their function and for immune homeostasis. Using intravital imaging, Chauveau et al. reveal that a network of perivascular pathways supports T cell migration into splenic T zones and describe a dynamic multi-step cascade of entry.

During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments(1–3). These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell(4,5). Most mesenchymal and epithelial cells and some, but not all, cancer cells actively generate their migratory path using pericellular tissue proteolysis(6). By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion(7), raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes, and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus, which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore, cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted, migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration, our findings link the fundamental organization of cellular polarity to the strategy of locomotion.

Dendritic cell (DC) actin dynamics at the immunological synapse with T cells is partially regulated by the WAVE regulatory complex. Absence of DC WAVE leads to reduced actin dynamics, prolonged cell–cell contacts, and impaired T cell priming., Dendritic cells (DCs) are crucial for the priming of naive T cells and the initiation of adaptive immunity. Priming is initiated at a heterologous cell–cell contact, the immunological synapse (IS). While it is established that F-actin dynamics regulates signaling at the T cell side of the contact, little is known about the cytoskeletal contribution on the DC side. Here, we show that the DC actin cytoskeleton is decisive for the formation of a multifocal synaptic structure, which correlates with T cell priming efficiency. DC actin at the IS appears in transient foci that are dynamized by the WAVE regulatory complex (WRC). The absence of the WRC in DCs leads to stabilized contacts with T cells, caused by an increase in ICAM1-integrin–mediated cell–cell adhesion. This results in lower numbers of activated and proliferating T cells, demonstrating an important role for DC actin in the regulation of immune synapse functionality.

Cell production and differentiation for the acquisition of specific functions are key features of living systems. The dynamic network of cellular microtubules provides the necessary platform to accommodate processes associated with the transition of cells through the individual phases of cytogenesis. Here, we show that the plant hormone cytokinin fine‐tunes the activity of the microtubular cytoskeleton during cell differentiation and counteracts microtubular rearrangements driven by the hormone auxin. The endogenous upward gradient of cytokinin activity along the longitudinal growth axis in Arabidopsis thaliana roots correlates with robust rearrangements of the microtubule cytoskeleton in epidermal cells progressing from the proliferative to the differentiation stage. Controlled increases in cytokinin activity result in premature re‐organization of the microtubule network from transversal to an oblique disposition in cells prior to their differentiation, whereas attenuated hormone perception delays cytoskeleton conversion into a configuration typical for differentiated cells. Intriguingly, cytokinin can interfere with microtubules also in animal cells, such as leukocytes, suggesting that a cytokinin‐sensitive control pathway for the microtubular cytoskeleton may be at least partially conserved between plant and animal cells., The plant hormone cytokinin can regulate microtubule orientation and polymerization in the plant root epidermis as well as in mammalian leukocytes, indicating a conserved mode of action.

Dendritic cells (DCs) are crucial for the priming of naïve T cells and the initiation of adaptive immunity. Priming is initiated at a heterologous cell-cell contact, the immunological synapse (IS). While it is established that actin dynamics regulates signalling at the T cell side of the contact, little is known about the cytoskeletal contribution on the DC side. We show that that the DC cytoskeleton is decisive for the formation of a multifocal synaptic structure, which correlates with T cell priming efficiency. We demonstrate that DC actin appears in transient foci at the IS and that these foci are dynamized by the WAVE complex. Absence of WAVE in DCs leads to stabilized contacts with T cells, caused by an increase in ICAM1-integrin mediated cell-cell adhesions. This results in a lower number of activated and proliferating T cells. Our results reveal an important role of DC actin in the regulation of synaptic contacts with crucial relevance for full T cell expansion.

Cells navigating through tissues face a fundamental challenge: while multiple cellular protrusions explore different paths through the complex geometry of an interstitial matrix the cell needs to avoid becoming too long or ramified, which might ultimately lead to a loss of physical coherence. How a cell surveys its own shape to inform the actomyosin system to retract entangled or stretched protrusions is not understood. Here, we demonstrate that spatially distinct microtubule (MT) dynamics regulate amoeboid cell migration by locally specifying the retraction of explorative protrusions. In migrating dendritic cells (DCs), the microtubule organizing center (MTOC) guides the path through a three dimensional (3D) interstitium and local MT depolymerization in protrusions remote from the MTOC triggers myosin II dependent contractility via the RhoA exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: i) impaired cell edge coordination during path-finding and ii) defective adhesion-resolution. Such compromised cell shape control is particularly hindering when cells navigate through geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. Our data demonstrate that MTs control cell shape and coherence by locally controlling protrusion-retraction dynamics of the actomyosin system.

The WAVE regulatory complex (WRC) is crucial for assembly of the peripheral branched actin network constituting one of the main drivers of eukaryotic cell migration. Here, we uncover an essential role of the hematopoietic-specific WRC component HEM1 for immune cell development. Germline-encoded HEM1 deficiency underlies an inborn error of immunity with systemic autoimmunity, at cellular level marked by WRC destabilization, reduced filamentous actin and failure to assemble lamellipodia. Hem1(-/-) mice display systemic autoimmunity, phenocopying the human disease. In the absence of Hem1, B cells become deprived of extracellular stimuli necessary to maintain the strength of B-cell receptor signaling at a level permissive for survival of non-autoreactive B cells. This shifts the balance of B-cell fate choices towards autoreactive B cells and thus autoimmunity.

An alternate route for metastatic cells Metastatic tumor cells are thought to reach distant organs by traveling through the blood circulation or the lymphatic system. Two studies of mouse models now suggest a hybrid route for tumor cell dissemination. Pereira et al. and Brown et al. used distinct methodologies to monitor the fate of tumor cells in lymph nodes. They found that tumor cells could invade local blood vessels within a node, exit the node by entering the blood circulation, then go on to colonize the lung. Whether this dissemination route occurs in cancer patients is unknown; the answer could potentially change the way that affected lymph nodes are treated in cancer. Science , this issue p. 1403 , p. 1408

IgM provides early protection against pathogens. How IgM is exported out of lymph nodes remains unknown. Thierry et al. report that B cells utilize a system of paracortical conduits to rapidly export their IgM to the periphery., Immunoglobulin M (IgM) is the first type of antibody produced during acute infections and thus provides an early line of specific defense against pathogens. Being produced in secondary lymphoid organs, IgM must rapidly be exported to the blood circulation. However, it is currently unknown how such large pentameric molecules are released from lymph nodes (LNs). Here, we show that upon immunization, IgM transiently gains access to the luminal side of the conduit system, a reticular infrastructure enabling fast delivery of tissue-derived soluble substances to the LN parenchyma. Using microinjections of purified IgM, we demonstrate that conduit-associated IgM is delivered by neither the afferent lymph nor the blood, but is locally conveyed by conduits. Exploiting in vivo models, we further demonstrate that conduit-associated IgM is locally and transiently produced by activated, antigen-specific B cells migrating in the T cell zone. Thus, our study reveals that the conduit system is coopted by B cells to rapidly export secreted IgM out of LNs., Graphical Abstract

During neural tube closure and spinal cord development, many cells die in both the central and peripheral nervous systems (CNS and PNS, respectively). However, myeloid-derived professional phagocytes have not yet colonized the trunk region during early neurogenesis. How apoptotic cells are removed from this region during these stages remains largely unknown. Using live imaging in zebrafish, we demonstrate that neural crest cells (NCC) respond rapidly to dying cells and phagocytose cellular debris around the neural tube. Additionally, NCCs have the ability to enter the CNS through motor exit point transition zones and clear debris in the spinal cord. Surprisingly, NCC phagocytosis mechanistically resembles macrophage phagocytosis and their recruitment towards cellular debris is mediated by interleukin-1β. Taken together, our results reveal a role for NCCs in phagocytosis of debris in the developing nervous system before the presence of professional phagocytes.

To induce adaptive immunity, dendritic cells (DCs) migrate through afferent lymphatic vessels (LVs) to draining lymph nodes (dLNs). This process occurs in several consecutive steps. Upon entry into lymphatic capillaries, DCs first actively crawl into downstream collecting vessels. From there, they are next passively and rapidly transported to the dLN by lymph flow. Here, we describe a role for the chemokine CCL21 in intralymphatic DC crawling. Performing time-lapse imaging in murine skin, we found that blockade of CCL21—but not the absence of lymph flow—completely abolished DC migration from capillaries toward collecting vessels and reduced the ability of intralymphatic DCs to emigrate from skin. Moreover, we found that in vitro low laminar flow established a CCL21 gradient along lymphatic endothelial monolayers, thereby inducing downstream-directed DC migration. These findings reveal a role for intralymphatic CCL21 in promoting DC trafficking to dLNs, through the formation of a flow-induced gradient., Cell Reports, 14 (7), ISSN:2666-3864, ISSN:2211-1247

A chemokine's sugary release As immune cells survey the body for pathogens, they circulate through the blood and migrate through the lymphatic system. The latter route allows for tissues and lymph nodes—the central hubs of the immune system—to communicate. Kiermaier et al. reveal the importance of the monosaccharide sialic acid in keeping immune cells in motion. Multiple sialic acids decorate the surface CCR7 on immune cells. CCR7 recognizes proteins called chemokines, which direct where cells move in the body. Sialic acids on CCR7 release one such chemokine present on lymph node endothelial cells from an inhibited state, allowing immune cells to enter lymph nodes. Science , this issue p. 186

Moalli et al. combine in vitro CD8+ T cell motility analysis with intravital imaging of mouse tissues to identify the actomyosin regulator Myo9b as a central player for nonlymphoid tissue infiltration during adaptive immune responses by facilitating crossing of tissue barriers., T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b−/− CD8+ T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b−/− CD8+ T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b−/− CD8+ T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8+ T cell adaptation from lymphoid to NLT surveillance and the establishment of protective tissue–resident T cell populations.

Chemokines are secreted proteins that regulate a range of processes in eukaryotic organisms. Interestingly, different chemokine receptors control distinct biological processes, and the same receptor can direct different cellular responses, but the basis for this phenomenon is not known. To understand this property of chemokine signaling, we examined the function of the chemokine receptors Cxcr4a, Cxcr4b, Ccr7, Ccr9 in the context of diverse processes in embryonic development in zebrafish. Our results reveal that the specific response to chemokine signaling is dictated by cell-type-specific chemokine receptor signal interpretation modules (CRIM) rather than by chemokine-receptor-specific signals. Thus, a generic signal provided by different receptors leads to discrete responses that depend on the specific identity of the cell that receives the signal. We present the implications of employing generic signals in different contexts such as gastrulation, axis specification and single-cell migration., eLife digest Every process in the body is regulated by a complex network of interactions between different molecules and cells. Chemokines, for example, are tiny molecules produced by a cell that are involved in a range of processes, from development to immune responses and cancer. When chemokines bind to a specific protein on another cell, called the chemokine receptor, it stimulates different signaling pathways inside the cell. Consequently, chemokine receptors are equally important for regulating processes as diverse as the movement of cells during development and growth, or activating immune responses. Mammals have over 20 different chemokine receptors, and the same receptor can have different roles depending in which cell type it is found in. For example, in one cell type it may stimulate an action such as cell growth, but in another, it may block this process. Until now, it was unclear how chemokine receptors can achieve such different effects. One theory was that chemokine receptors initiate a distinct signaling cascade, a phenomenon termed ‘signaling bias’, depending on the type of chemokine or receptor. Here, Malhotra et al. used zebrafish embryos to investigate how four specific chemokine receptors regulate different events during early development. They found that the same chemokine receptor could direct different reactions in distinct cell types, while different receptors could also cause the same response in a specific cell type. In other words, the effect of a chemokine receptor depends on the cell type rather than the type of receptor. Since each of these receptors was able to control processes that it normally does not regulate in other cells, Malhotra et al. suggest that different chemokine receptors provide the same generic signal when activated, which the specific cell types then interpret accordingly. A next step will be to test how other chemokine receptors behave in different contexts, for example during an immune response. If the receptors work on the same principle regardless of the process, it could help to explain why faulty expression of chemokine receptors play such an important role during development and in disease. It could further highlight why blocking one receptor may not have any consequences, as they are dispensable and can be replaced by other receptors in the cell.

Inflammation stimulates lymphatic endothelial cells to release exosomes, which accumulate in the perivascular stroma. Brown et al. show that these exosomes promote the directional migration of dendritic cells along guidance cues in complex environments by enhancing dynamic cellular protrusions in a CX3CL1-dependent manner., Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments.

Although much is known about the physiological framework of T cell motility, and numerous rate-limiting molecules have been identified through loss-of-function approaches, an integrated functional concept of T cell motility is lacking. Here, we used in vivo precision morphometry together with analysis of cytoskeletal dynamics in vitro to deconstruct the basic mechanisms of T cell migration within lymphatic organs. We show that the contributions of the integrin LFA-1 and the chemokine receptor CCR7 are complementary rather than positioned in a linear pathway, as they are during leukocyte extravasation from the blood vasculature. Our data demonstrate that CCR7 controls cortical actin flows, whereas integrins mediate substrate friction that is sufficient to drive locomotion in the absence of considerable surface adhesions and plasma membrane flux.

Replication-deficient recombinant adenoviruses are potent vectors for the efficient transient expression of exogenous genes in resting immune cells. However, most leukocytes are refractory to efficient adenoviral transduction as they lack expression of the coxsackie/adenovirus receptor (CAR). To circumvent this obstacle, we generated the R26/CAG-CARΔ1(StopF) (where R26 is ROSA26 and CAG is CMV early enhancer/chicken β actin promoter) knock-in mouse line. This strain allows monitoring of in situ Cre recombinase activity through expression of CARΔ1. Simultaneously, CARΔ1 expression permits selective and highly efficient adenoviral transduction of immune cell populations, such as mast cells or T cells, directly ex vivo in bulk cultures without prior cell purification or activation. Furthermore, we show that CARΔ1 expression dramatically improves adenoviral infection of in vitro differentiated conventional and plasmacytoid dendritic cells (DCs), basophils, mast cells, as well as Hoxb8-immortalized hematopoietic progenitor cells. This novel dual function mouse strain will hence be a valuable tool to rapidly dissect the function of specific genes in leukocyte physiology.

International audience; Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.

Stromal cells in the subcapsular sinus of the lymph node 'decide' which cells and molecules are allowed access to the deeper parenchyma. The glycoprotein PLVAP is a crucial component of this selector function.

Cell migration is central to a multitude of physiological processes, including embryonic development, immune surveillance, and wound healing, and deregulated migration is key to cancer dissemination. Decades of investigations have uncovered many of the molecular and physical mechanisms underlying cell migration. Together with protrusion extension and cell body retraction, adhesion to the substrate via specific focal adhesion points has long been considered an essential step in cell migration. Although this is true for cells moving on two-dimensional substrates, recent studies have demonstrated that focal adhesions are not required for cells moving in three dimensions, in which confinement is sufficient to maintain a cell in contact with its substrate. Here, we review the investigations that have led to challenging the requirement of specific adhesions for migration, discuss the physical mechanisms proposed for cell body translocation during focal adhesion–independent migration, and highlight the remaining open questions for the future.

Cell migration has important functions in immune response, development and cancer proliferation. Cell movement essentially requires the establishment of an axis of polarity. To maintain migration in one direction - called persistent cell migration - the polarity axis needs to be stabilized in the direction of movement.Biochemical feedback loops between signaling molecules have been recognized to play an important role for stabilizing cell polarity by reinforcing actin polymerization at the leading edge. Here we describe a reverse feedback mechanism coupling the retrograde flowing actin cytoskeleton in migrating cells back to cell polarity and persistent migration supporting an essential mechano-chemical coupling of signaling cascades and the flowing cytoskeletal meshwork in migrating cells (Maiuri et al., 2015). We used genetic, physical and chemical tools to modulate cytoskeletal flows in migrating dendritic cells and found a strong increase in the stability of cellular polarization and migration persistence upon increasing retrograde flow speed. We provide experimental evidence that the spatial distribution of different actin binding proteins (Myosin-GFP, Lifeact-GFP and Utrophin-GFP) with varying actin-binding strength gets significantly shifted towards the cell rear for increasing retrograde flow speeds upon sufficient coupling to the flowing actin network. Finally we show that a minimal theoretical model build on the mechano-chemical coupling between polarity markers and cytoskeletal flows can predict the stability of cell polarity in dependence on retrograde flow speeds and can capture the broad range of cell migration phenotypes from Brownian, to persistent and intermittent random walks.Maiuri, P.∗, Rupprecht, J.-F.∗, Wieser, S.∗, Ruprecht, V., Benichou, O., Carpi, N., Coppey, M., De Beco, S., Gov, N., Heisenberg, C.-P., et al. (2015). Actin Flows Mediate a Universal Coupling between Cell Speed and Cell Persistence. Cell 161, 374-386. ∗contributed equally.

SummaryLittle is known about mechanisms determining the homeostasis of lymphocytes within lymphoid organs. Applying different mouse models, including conditionally proficient Ccr7 gene-targeted mice, we now show that semimature steady state dendritic cells (sDCs) constitutively trafficking into lymph nodes (LNs) were essential contributors to T cell homeostasis in these organs. sDCs provided vascular endothelial growth factor known to support high endothelial venule formation, thus facilitating enhanced homing of T cells to LNs. The presence of sDCs led to increased CCL21 production in T-zone fibroblastic reticular cells. CCL21 is a ligand for CCR7 known to regulate homing as well as retention of T cells in LNs. In addition, we provide evidence that CCL21 binds to the surface of DCs via its heparin-binding domain, further explaining why T cells leave LNs more rapidly in the absence of sDCs. Together, these data reveal multiple roles for sDCs in regulating T cell homeostasis in LNs.

In this issue of Cell, Skau et al. show that the formin FMN2 organizes a perinuclear actin cytoskeleton that protects the nucleus and its genomic content of migrating cells squeezing through small spaces.

CD4+CD25+ regulatory T cell (Treg) entry into secondary lymphoid organs and local expansion is critical for their immunosuppressive function. Long-term application of the sphingosine-1 phosphate receptor agonist FTY720 exerts pleiotropic anti-inflammatory effects, whereas short-term FTY720 boosts antiviral immunity. In this study, we provide evidence that FTY720 potently inhibits Treg proliferation in vitro and in vivo without affecting their viability, phenotype, or in vitro immunosuppression. In contrast, adoptively transferred Treg exposed ex vivo to FTY720 lost their protective effects in murine models of acute glomerulonephritis and acute graft-vs-host disease. On a cellular level, FTY720 inhibits IL-2-induced STAT-5 phosphorylation, paralleled by a loss of FoxP3 expression during Treg expansion in vitro. Notably, loss of in vivo immunosuppression is not due to impaired migration to or localization within secondary lymphoid organs. We could even show a selective trapping of adoptively transferred Treg in inflammatory lymph nodes by FTY720. Finally, Treg isolated from animals systemically exposed to FTY720 also exhibit a significantly impaired proliferative response upon restimulation when compared with Treg isolated from solvent-treated animals. In summary, our data suggest that sphingosine-1 phosphate receptor-mediated signals induced by FTY720 abrogate their in vivo immunosuppressive potential by blocking IL-2 induced expansion, which is indispensable for their in vivo immunosuppressive activity.

Kindlin-3 interacts with β1 and β3 integrins on platelets, and Kindlin-3–deficient mice have defects in platelet activation and blood clotting. Moser et al. now show that these mice also have defects in β2 integrin activation on leukocytes, leading to severely compromised leukocyte adhesion to the endothelium. The combined platelet and leukocyte defects of these mice resemble those seen in individuals with the leukocyte adhesion deficiency syndrome LAD-III. Other papers in this issue by Malinin et al. and Svensson et al. provide evidence that KINDLIN-3 dysfunction does indeed underlie this type of human disease syndrome (pages 249–250, 306–312 and 313–318).

Although both processes occur at similar rates, leukocyte extravasation from the blood circulation is well investigated, whereas intravasation into lymphatic vessels has hardly been studied. In contrast to a common assumption—that intra- and extravasation follow similar molecular principles—we previously showed that lymphatic entry of dendritic cells (DCs) does not require integrin-mediated adhesive interactions. In this study, we demonstrate that DC-entry is also independent of pericellular proteolysis, raising the question of whether lymphatic vessels offer preexisting entry routes. We find that the perilymphatic basement membrane of initial lymphatic vessels is discontinuous and therefore leaves gaps for entering cells. Using a newly developed in situ live cell imaging approach that allows us to dynamically visualize the cells and their extracellular environment, we demonstrate that DCs enter through these discontinuities, which are transiently mechanically dilated by the passaging cells. We further show that penetration of the underlying lymphatic endothelial layer occurs through flap valves lacking continuous intercellular junctions. Together, we demonstrate free cellular communication between interstitium and lymphatic lumen.

Lymphocyte motility in lymph nodes is regulated by chemokines, but the contribution of integrins to this motility remains obscure. Here we examined lymphocyte migration over CCR7-binding chemokines that 'decorate' lymph node stroma. In a shear-free environment, surface-bound lymph node chemokines but not their soluble counterparts promoted robust and sustained T lymphocyte motility. The chemokine CCL21 induced compartmentalized clustering of the integrins LFA-1 and VLA-4 in motile lymphocytes, but both integrins remained nonadhesive to ligands on lymphocytes, dendritic cells and stroma. The application of shear stress to lymphocytes interacting with CCL21 and integrin ligands promoted robust integrin-mediated adhesion. Thus, lymph node chemokines that promote motility and strongly activate lymphocyte integrins under shear forces fail to stimulate stable integrin adhesiveness in extravascular shear-free environments.

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space., Dendritic cells alternate between fast and slow migratory behaviours, however in the absence of a component of the antigen processing machinery, migration is uniform and fast. Chabaud et al. now show that slow migration results from the relocalisation of myosin II to the cell front where it promotes antigen capture.

SummaryCell movement has essential functions in development, immunity, and cancer. Various cell migration patterns have been reported, but no general rule has emerged so far. Here, we show on the basis of experimental data in vitro and in vivo that cell persistence, which quantifies the straightness of trajectories, is robustly coupled to cell migration speed. We suggest that this universal coupling constitutes a generic law of cell migration, which originates in the advection of polarity cues by an actin cytoskeleton undergoing flows at the cellular scale. Our analysis relies on a theoretical model that we validate by measuring the persistence of cells upon modulation of actin flow speeds and upon optogenetic manipulation of the binding of an actin regulator to actin filaments. Beyond the quantitative prediction of the coupling, the model yields a generic phase diagram of cellular trajectories, which recapitulates the full range of observed migration patterns.

T cells develop in the thymus in a highly specialized cellular and extracellular microenvironment. The basement membrane molecule, laminin-5 (LN-5), is predominantly found in the medulla of the human thymic lobules. Using high-resolution light microscopy, we show here that LN-5 is localized in a bi-membranous conduit-like structure, together with other typical basement membrane components including collagen type IV, nidogen and perlecan. Other interstitial matrix components, such as fibrillin-1 or -2, tenascin-C or fibrillar collagen types, were also associated with these structures. Three-dimensional (3D) confocal microscopy suggested a tubular structure, whereas immunoelectron and transmission electron microscopy showed that the core of these tubes contained fibrillar collagens enwrapped by the LN-5-containing membrane. These medullary conduits are surrounded by thymic epithelial cells, which in vitro were found to bind LN-5, but also fibrillin and tenascin-C. Dendritic cells were also detected in close vicinity to the conduits. Both of these stromal cell types express major histocompatibility complex (MHC) class II molecules capable of antigen presentation. The conduits are connected to blood vessels but, with an average diameter of 2 μm, they are too small to transport cells. However, evidence is provided that smaller molecules such as a 10 kDa dextran, but not large molecules (>500 kDa), can be transported in the conduits. These results clearly demonstrate that a conduit system, which is also known from secondary lymphatic organs such as lymph nodes and spleen, is present in the medulla of the human thymus, and that it might serve to transport small blood-borne molecules or chemokines to defined locations within the medulla.

Objectives. The EGFR is expressed in malignant ovarian tumor tissue, and tissue content of EGFR has been directly associated with poor prognosis in patients with ovarian cancer. The uPA system plays a role in pericellular proteolysis, cell migration, invasion, and is over-expressed in ovarian cancer. This study explored the effects of EGF on uPAR expression in the ovarian cancer cell line OVCAR-3. Methods. We used OVCAR-3 cells and the following methods: cell migration assay, time-lapse video microscopy, real-time PCR, assays for cellular binding of I-125-uPA and cellular degradation of I-125-uPA:PAI-1 complex, biosynthetic labeling using S-35-methionin, Western blot, Northern blot, and ELISAs for uPA, PAI-1, and uPAR. Results. EGF up-regulates both protein and mRNA not only for uPAR, but also for the ligand uPA and its inhibitor PAI-1. Cell surface uPAR, in control as well as EGF-stimulated cells, is present only in the intact, not the cleaved, form. Ligand binding experiments showed an increase of endogenously occupied uPAR, whereas non-occupied receptor sites were not increased. In addition, EGF treatment resulted in decreased degradation of radiolabeled uPA:PAI-1 complex. This suggests decreased internalization of uPAR, since the complex is internalized together with uPAR. Like EGF, colchicine, which inhibits endocytosis, increased cell surface expression of uPAR. In addition, we found an immediate increase of uPAR after exposing the cells to EGF and this was accompanied by a transient increase of cell migration. The increase of cell surface uPAR in response to EGF is accompanied by increased release of the soluble form of uPAR (suPAR) to the medium as well as by increased cell migration. Both uPAR and suPAR increased in cells treated with the endocytosis inhibitor colchicine even though cell migration was inhibited, suggesting that the mechanism of uPAR shedding is not related to cell migration. Conclusion. Increased cell surface uPAR in response to EGF stimulation results from mobilization of uPAR from detergent-resistant domains, increased expression of uPAR mRNA, and decreased internalization and degradation of uPAR. Both the anti-uPAR antibody R3, which inhibits binding of uPA, and the EGFR phosphorylation inhibitor Iressa inhibited cell migration in response to uPA as well as to EGF, suggesting that EGFR and uPAR are engaged in the same multiprotein assembly on the cell surface. (C) 2005 Elsevier Inc. All rights reserved.

Resident dendritic cells (DC) within the T cell area of the lymph node take up soluble antigens that enter via the afferent lymphatics before antigen carrying DC arrive from the periphery. The reticular network within the lymph node is a conduit system forming the infrastructure for the fast delivery of soluble substances from the afferent lymph to the lumen of high endothelial venules (HEVs). Using high-resolution light microscopy and 3D reconstruction, we show here that these conduits are unique basement membrane-like structures ensheathed by fibroblastic reticular cells with occasional resident DC embedded within this cell layer. Conduit-associated DC are capable of taking up and processing soluble antigens transported within the conduits, whereas immigrated mature DC occur remote from the reticular fibers. The conduit system is, therefore, not a closed compartment that shuttles substances through the lymph node but represents the morphological equivalent to the filtering function of the lymph node.

An active involvement of blood–brain barrier endothelial cell basement membranes in development of inflammatory lesions in the central nervous system (CNS) has not been considered to date. Here we investigated the molecular composition and possible function of the extracellular matrix encountered by extravasating T lymphocytes during experimental autoimmune encephalomyelitis (EAE).Endothelial basement membranes contained laminin 8 (α4β1γ1) and/or 10 (α5β1γ1) and their expression was influenced by proinflammatory cytokines or angiostatic agents. T cells emigrating into the CNS during EAE encountered two biochemically distinct basement membranes, the endothelial (containing laminins 8 and 10) and the parenchymal (containing laminins 1 and 2) basement membranes. However, inflammatory cuffs occurred exclusively around endothelial basement membranes containing laminin 8, whereas in the presence of laminin 10 no infiltration was detectable. In vitro assays using encephalitogenic T cell lines revealed adhesion to laminins 8 and 10, whereas binding to laminins 1 and 2 could not be induced. Downregulation of integrin α6 on cerebral endothelium at sites of T cell infiltration, plus a high turnover of laminin 8 at these sites, suggested two possible roles for laminin 8 in the endothelial basement membrane: one at the level of the endothelial cells resulting in reduced adhesion and, thereby, increased penetrability of the monolayer; and secondly at the level of the T cells providing direct signals to the transmigrating cells.

A hallmark of immune cell trafficking is directional guidance via gradients of soluble or surface bound chemokines. Vascular endothelial cells produce, transport and deposit either their own chemokines or chemokines produced by the underlying stroma. Endothelial heparan sulfate (HS) was suggested to be a critical scaffold for these chemokine pools, but it is unclear how steep chemokine gradients are sustained between the lumenal and ablumenal aspects of blood vessels. Addressing this question by semi-quantitative immunostaining of HS moieties around blood vessels with a pan anti-HS IgM mAb, we found a striking HS enrichment in the basal lamina of resting and inflamed post capillary skin venules, as well as in high endothelial venules (HEVs) of lymph nodes. Staining of skin vessels with a glycocalyx probe further suggested that their lumenal glycocalyx contains much lower HS density than their basolateral extracellular matrix (ECM). This polarized HS pattern was observed also in isolated resting and inflamed microvascular dermal cells. Notably, progressive skin inflammation resulted in massive ECM deposition and in further HS enrichment around skin post capillary venules and their associated pericytes. Inflammation-dependent HS enrichment was not compromised in mice deficient in the main HS degrading enzyme, heparanase. Our results suggest that the blood vasculature patterns steep gradients of HS scaffolds between their lumenal and basolateral endothelial aspects, and that inflammatory processes can further enrich the HS content nearby inflamed vessels. We propose that chemokine gradients between the lumenal and ablumenal sides of vessels could be favored by these sharp HS scaffold gradients.

During inflammation, lymph nodes swell with an influx of immune cells. New findings identify a signalling pathway that induces relaxation in the contractile cells that give structure to these organs. See Letter p.498 Lymph nodes are dynamic structures that must respond rapidly to large cellular influxes provoked by local inflammation. However, how the lymph node stromal compartment reacts to accommodate lymph node expansion remains unclear. This study shows that the lymph node stromal compartment can accommodate large numbers of infiltrating lymphocytes by relaxing the cytoskeleton of fibroblastic reticular cells, allowing the cells to stretch and the lymph node to expand. This lymph node remodelling reaction is driven by the interaction of CLEC-2 protein on incoming antigen-presenting dendritic cells with podoplanin on fibroblastic reticular cells.

Langerhans cells (LCs) are a unique subset of dendritic cells (DCs) that express epithelial adhesion molecules, allowing them to form contacts with epithelial cells and reside in epidermal/epithelial tissues. The dynamic regulation of epithelial adhesion plays a decisive role in the life cycle of LCs. It controls whether LCs remain immature and sessile within the epidermis or mature and egress to initiate immune responses. So far, the molecular machinery regulating epithelial adhesion molecules during LC maturation remains elusive. Here, we generated pure populations of immature human LCs in vitro to systematically probe for gene-expression changes during LC maturation. LCs down-regulate a set of epithelial genes including E-cadherin, while they upregulate the mesenchymal marker N-cadherin known to facilitate cell migration. In addition, N-cadherin is constitutively expressed by monocyte-derived DCs known to exhibit characteristics of both inflammatory-type and interstitial/dermal DCs. Moreover, the transcription factors ZEB1 and ZEB2 (ZEB is zinc-finger E-box-binding homeobox) are upregulated in migratory LCs. ZEB1 and ZEB2 have been shown to induce epithelial-to-mesenchymal transition (EMT) and invasive behavior in cancer cells undergoing metastasis. Our results provide the first hint that the molecular EMT machinery might facilitate LC mobilization. Moreover, our study suggests that N-cadherin plays a role during DC migration.

Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p160 coiled-coil kinase (ROCK)-myosin II-mediated uropod contractility is required for integrin de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonactivated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T cell migration through narrow, but not large, pores in vitro. T cell crawling on chemokine-coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated T cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod contraction during lymphocyte recirculation.

The Minisymposium “Cell Migration and Motility” was attended by approximately 500 visitors and covered a broad range of questions in the field using diverse model systems. Topics comprised actin dynamics, cell polarity, force transduction, signal transduction, barrier transmigration, and chemotactic guidance. The session started with the 2010 American Society for Cell Biology Early Career Life Science awardee, Anna Kashina (University of Pennsylvania), who reported on the role of arginylation in the dynamics of lamellipodial actin. In contrast to γ-actin, β-actin preferentially localizes to the leading edge of migrating cells and is arginylated. Surprisingly, the reason for this differential arginylation is not inferred by differences at the amino acid level but by different mRNA codon usage that results in faster β-actin translation and protein folding kinetics, which in turn affect arginylation, protein stability, and turnover rates. The talk was followed by a presentation on the chemotropic response of budding yeast—the basis of mate recognition. Jayme Johnson (Duke University) showed data supporting a new model in which the “polarity patch,” a polarity complex that is condensed by positive feedback and directs polarized growth, searches the inner membrane for active receptors. Local, receptor-mediated signaling slows patch motility and thereby biases the growth response toward the pheromone gradient. Cochair Carole Parent (National Cancer Institute, National Institutes of Health) then reported on a new chemotactic signalling pathway in neutrophil granulocytes that shows partial homology with attractant sensing in Dictyostelium. The pathway involves a mammalian target of rapamycin complex 2, protein kinase C, and cyclic adenosine monophosphate–dependent signalling cascade that regulates myosin II–dependent contractility at the trailing edge of the cell and thereby balances leading-edge protrusion with trailing-edge retraction during directed motility. Daria Siekhaus (Skirball Institute, New York University) followed with a presentation on Drosophila immune cell distribution during early embryogenesis. The path of these cells involves the penetration of an epithelial layer, and a genetic screen revealed that, in analogy to the leukocyte extravasation process in vertebrates, this step involves inside-out signalling of integrins, while migration through the rest of the interstitium is independent of these receptors. Integrin activation is mediated by the guanosine 5′-triphosphate–binding proteins Rap1 and RhoL, and the weakening of epithelial junctions is sufficient to rescue the immune cell autonomous penetration defects. In the next presentation cochair Michael Sixt (Institute of Science and Technology, Austria) presented data supporting the idea that the presentation of guidance cues determines the migratory mode of leukocytes. When dendritic cells are exposed to soluble chemokines, they migrate in a nonadhesive manner, and consequently the extracellular substrate does not play instructive roles for cell guidance. When the chemokine is immobilized to surfaces, integrin activation is triggered, the cells become adhesive, and they migrate along the chemokine-decorated matrix structure. The session concluded with a talk by Matthew Paszek (University of California, San Francisco), who proposed a new role of the glycocalyx in cell adhesion. A combination of immunofluorescence and fluorescence interference contrast microscopy demonstrated that at sites of high glycoprotein density the plasma membrane of adherent cells is elevated from the substrate, while the spatially separated adhesion sites are in close proximity to the substrate. Consequently, enhanced glycoprotein expression causes irregular membrane topography and imposes a vertical pull on integrin receptors, causing enhanced force coupling and adhesion site maturation.

The shuttling of leukocytes between the bloodstream and interstitial tissues involves different locomotion strategies that are governed by locally presented soluble and cell-bound signals. Recent studies have furthered our understanding of the rapidly advancing field of leukocyte migration, particularly regarding cellular and subcellular events at the level of the venular wall. Furthermore, emerging cellular models are now addressing the transition from an adherent mode to a non-adherent state, incorporating mechanisms that support an efficient migratory profile of leukocytes in the interstitial tissue beyond the venular wall.

The mammalian actin-binding protein 1 (mAbp1, Hip-55, SH3P7) is phosphorylated by the nonreceptor tyrosine kinase Syk that has a fundamental effect for several β2 integrin (CD11/CD18)–mediated neutrophil functions. Live cell imaging showed a dynamic enrichment of enhanced green fluorescence protein–tagged mAbp1 at the phagocytic cup of neutrophil-like differentiated HL-60 cells during β2 integrin–mediated phagocytosis of serum-opsonized Escherichia coli. The genetic absence of Syk or its pharmacologic inhibition using piceatannol abrogated the proper localization of mAbp1 at the phagocytic cup. The genetic absence or down-regulation of mAbp1 using the RNA interference technique significantly compromised β2 integrin–mediated phagocytosis of serum-opsonized E coli or Salmonella typhimurium in vitro as well as clearance of S typhimurium infection in vivo. Moreover, the genetic absence of mAbp1 almost completely abrogated firm neutrophil adhesion under physiologic shear stress conditions in vitro as well as leukocyte adhesion and extravasation in inflamed cremaster muscle venules of mice treated with tumor-necrosis factor α. Functional analysis showed that the down-regulation of mAbp1 diminished the number of β2 integrin clusters in the high-affinity conformation under flow conditions. These unanticipated results define mAbp1 as a novel molecular player in integrin biology that is critical for phagocytosis and firm neutrophil adhesion under flow conditions.

Langerhans cells and dermal dendritic cells migrate to the draining lymph nodes through dermal lymphatic vessels. They do so in the steady-state and under inflammatory conditions. Peripheral T cell tolerance or T cell priming, respectively, are the consequences of migration. The nature of dendritic cell-containing vessels was mostly defined by electron microscopy or by their lack of blood endothelial markers. Selective markers for murine lymph endothelium were hitherto rare or not available. Here, we utilised recently developed antibodies against the murine hyaluronan receptor, LYVE-1, to study the lymph vessel network in mouse skin in more detail. In hairless skin from the ears, lymph vessels were spread out in a horizontal plane. They formed anastomoses, and they possessed frequent blind endings that were occasionally open. Lymph vessels were wider than blood vessels, which were identified by their strong CD31 expression. In body wall skin LYVE-1 reactive vessels did not extend laterally but they dived straight down into the deeper dermis. There, they are connected to each other and formed a network similar to ear skin. The number and width of lymph vessels did not grossly change upon inflammatory stimuli such as skin explant culture or tape stripping. There were also no marked changes in caliber in response to the TLR 7/8 ligand Imiquimod. Double-labelling experiments of cultured skin showed that most of the strongly cell surface MHC II-expressing (i.e. activated) dendritic cells were confined to the lymph vessels. Langerin/CD207(+) cells within this population appeared later than dermal dendritic cells, i.e. langerin-negative cells. Comparable results were obtained after stimulating the skin in vivo with the TLR 7/8 ligand Imiquimod or by tape stripping. In untreated skin (i.e. steady state) a few MHC II(+) and Langerin/CD207(+) cells, presumably migrating skin dendritic cells including epidermal Langerhans cells, were consistently observed within the lymph vessels. The novel antibody reagents may serve as important tools to further study the dendritic cell traffic in the skin under physiological conditions as well as in conditions of adoptive dendritic cell transfer in immunotherapy.

The priming of a T cell results from its physical interaction with a dendritic cell (DC) that presents the cognate antigenic peptide. The success rate of such interactions is extremely low, because the precursor frequency of a naive T cell recognizing a specific antigen is in the range of 1:105-106. To make this principle practicable, encounter frequencies between DCs and T cells are maximized within lymph nodes (LNs) that are compact immunological projections of the peripheral tissue they drain. But LNs are more than passive meeting places for DCs that immigrated from the tissue and lymphocytes that recirculated via the blood. The microanatomy of the LN stroma actively organizes the cellular encounters by providing preformed migration tracks that create dynamic but highly ordered movement patterns. LN architecture further acts as a sophisticated filtration system that sieves the incoming interstitial fluid at different levels and guarantees that immunologically relevant antigens are loaded on DCs or B cells while inert substances are channeled back into the blood circulation. This review focuses on the non-hematopoietic infrastructure of the lymph node. We describe the association between fibroblastic reticular cell, conduit, DC, and T cell as the essential functional unit of the T-cell cortex.

Until recently little information was available on the molecular details of the extracellular matrix (ECM) of secondary lymphoid tissues. There is now growing evidence that these ECMs are unique structures, combining characteristics of basement membranes and interstitial or fibrillar matrices, resulting in scaffolds that are strong and highly flexible and, in certain secondary lymphoid compartments, also forming conduit networks for rapid fluid transport. This review will address the structural characteristics of the ECM of the murine spleen and its potential role as an organizer of immune cell compartments, with reference to the lymph node where relevant.

At least eight of the twelve known members of the beta1 integrin family are expressed on hematopoietic cells. Among these, the VCAM-1 receptor alpha4beta1 has received most attention as a main factor mediating firm adhesion to the endothelium during blood cell extravasation. Therapeutic trials are ongoing into the use of antibodies and small molecule inhibitors to target this interaction and hence obtain anti-inflammatory effects. However, extravasation is only one possible process that is mediated by beta1 integrins and there is evidence that they also mediate leukocyte retention and positioning in the tissue, lymphocyte activation and possibly migration within the interstitium. Genetic mouse models where integrins are selectively deleted on blood cells have been used to investigate these functions and further studies will be invaluable to critically evaluate therapeutic trials.

Integrins regulate cell behavior through the assembly of multiprotein complexes at the site of cell adhesion. Parvins are components of such a multiprotein complex. They consist of three members (alpha-, beta-, and gamma-parvin), form a functional complex with integrin-linked kinase (ILK) and PINCH, and link integrins to the actin cytoskeleton. Whereas alpha- and beta-parvins are widely expressed, gamma-parvin has been reported to be expressed in hematopoietic organs. In the present study, we report the expression pattern of the parvins in hematopoietic cells and the phenotypic analysis of gamma-parvin-deficient mice. Whereas alpha-parvin is not expressed in hematopoietic cells, beta-parvin is only found in myeloid cells and gamma-parvin is present in both cells of the myeloid and lymphoid lineages, where it binds ILK. Surprisingly, loss of gamma-parvin expression had no effect on blood cell differentiation, proliferation, and survival and no consequence for the T-cell-dependent antibody response and lymphocyte and dendritic cell migration. These data indicate that despite the high expression of gamma-parvin in hematopoietic cells it must play a more subtle role for blood cell homeostasis.

TNF-α has been clearly identified as central mediator of T cell activation-induced acute hepatic injury in mice, e.g., Con A hepatitis. In this model, liver injury depends on both TNFRs, i.e., the 55-kDa TNFR1 as well as the 75-kDa TNFR2. We show in this report that the hepatic TNFRs are not transcriptionally regulated, but are regulated by receptor shedding. TNF directly mediates hepatocellular death by activation of TNFR1 but also induces the expression of inflammatory proteins, such as cytokines and adhesion molecules. Here we provide evidence that resistance of TNFR1−/− and TNFR2−/− mice against Con A hepatitis is not due to an impaired production of the central mediators TNF and IFN-γ. Con A injection results in a massive induction of ICAM-1, VCAM-1, and E-selectin in the liver. Lack of either one of both TNFRs did not change adhesion molecule expression in the livers of Con A-treated mice, presumably reflecting the fact that other endothelial cell-activating cytokines up-regulated adhesion molecule expression. However, treatment of TNFR1−/− and TNFR2−/− mice with murine rTNF revealed a predominant role for TNFR1 for the induction of hepatic adhesion molecule expression. Pretreatment with blocking Abs against E- and P-selectin or of ICAM−/− mice with anti-VCAM-1 Abs failed to prevent Con A hepatitis, although accumulation of the critical cell population, i.e., CD4+ T cells was significantly inhibited. Hence, up-regulation of adhesion molecules during acute hepatitis unlikely contributes to organ injury but rather represents a defense mechanism.