Leukocyte migration in the interstitial space of non-lymphoid organs.

Leukocyte migration in the interstitial space of non-lymphoid organs Wolfgang Weninger1–3, Maté Biro1,4 and Rohit Jain1

Abstract | Leukocyte migration through interstitial tissues is essential for mounting a successful immune response. Interstitial motility is governed by a vast array of cell-intrinsic and cell-extrinsic factors that together ensure the proper positioning of immune cells in the context of specific microenvironments. Recent advances in imaging modalities, in particular intravital confocal and multi-photon microscopy, have helped to expand our understanding of the cellular and molecular mechanisms that underlie leukocyte navigation in the extravascular space. In this Review, we discuss the key factors that regulate leukocyte motility within three-dimensional environments, with a focus on neutrophils and T cells in non-lymphoid organs.

Centenary Institute for Cancer Medicine and Cell Biology, Newtown, New South Wales 2042, Australia. 2 Discipline of Dermatology, University of Sydney, New South Wales 2006, Australia. 3 Department of Dermatology, Royal Prince Alfred Hospital, Camperdown, New South Wales 2050, Australia. 4 Sydney Medical School, The University of Sydney, New South Wales 2006, Australia. Correspondence to W.W. e‑mail: w.weninger@ centenary.org.au doi:10.1038/nri3641 Published online 7 March 2014 1

Peripheral tissues, including the skin, gastrointestinal tract and lungs, are continuously exposed to a large number of noxious environmental agents, physical injuries and potentially harmful microorganisms or transformed cells. The immune system has specialized cells that can counter these attacks. Under homeostatic conditions, non-lymphoid organs are populated relatively sparsely with immune sentinels that can sense pathogen intrusion and danger signals. Upon activation, these cells alert immune effector cells, such as neutrophils, monocytes and T cells, which then enter organs from the bloodstream. Central to the functioning of this ‘alarm–recruit’ system is the migratory capacity of leukocytes, which must be able to rapidly reach any microenvironment within the body whenever called upon. Several prerequisites must be met for blood-borne cells to reach a target site. Initially, they need to adhere to the activated endothelium in postcapillary venules; a process that is governed by the multistep adhesion cascade1,2. This cascade consists of selectin-mediated tethering and rolling, firm adherence facilitated by activated integrins, and transendothelial migration (TEM). Once in the extravascular space, leukocytes need to negotiate a complex set of cell-intrinsic and cell-extrinsic factors that together contribute to their organized navigation through the interstitium. Such regulatory cues determine cell shape control, sensing and interpretation of chemoattractive and chemorepulsive gradients, forward propulsion mediated by the actomyosin cytoskeleton, and interactions with cells — as well as non-cellular structures — within tissue compartments. These processes ensure the timely arrival of leukocytes at damaged

or infected foci, where they exert their effector functions. Interstitial leukocyte navigation is important for the successful immunosurveillance of peripheral tissues, tissue repair, the clearance of pathogens and the eradication of tumour cells. Various model systems have been exploited for studying the mechanisms and dynamics of leukocyte migration. Two-dimensional and three-dimensional cell culture systems have greatly contributed to unravelling the factors that mediate cell shape control and directional migration. More recently, advances in intravital microscopic imaging approaches, in particular confocal and multi-photon microscopy in rodent and zebrafish models, have enhanced our knowledge of realtime leukocyte behaviour in the interstitium3–8. This Review examines recent progress in deciphering the mechanisms that regulate leukocyte motility in intact non-lymphoid organs from molecular, cellular and cell population perspectives. Emphasis will be on the migratory properties of neutrophils and T cells, which are among the most well-studied leukocyte subsets in this context. We discuss the lessons learnt from visualizing leukocyte behaviour in vivo, thereby providing a timely conceptual overview of interstitial immune cell navigation. The behaviour of various innate immune cell populations (other than neutrophils) has also been tracked in both steady-state and inflamed organs. From these investigations, it is apparent that innate immune cells are not merely passive tissue residents, but rather that they actively patrol their immediate microenvironments. Results from these studies are summarized in TABLE 1 and not further discussed.

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REVIEWS Table 1 | Intravital imaging studies of innate immune cell behaviour during tissue surveillance in peripheral organs Cell type

Tissue context

Cellular behaviour

Proposed biological function

Refs

Normal epidermis

LC bodies are immobile, but cells show occasional extensions and retractions of dendrites (dSEARCH)

Immunosurveillance

121,122

Inflamed epidermis

Lateral movement of LCs and increased motility of dendrites. Dendrites extend through tight junctions underneath the stratum corneum coupled to increased endocytic activity and antigen uptake

Immunosurveillance and initiation of immune responses

121,123

Normal dermis

DDCs migrate constitutively throughout the superficial dermis

Immunosurveillance

122

Dermal L. major infection

Migratory stop of DDCs concomitant with parasite uptake through motile cell processes

Recognition of pathogens and initiation of immune response

122

Normal small intestine

Transepithelial extensions of motile DC processes capable of catching bacteria in the gut lumen

Immunosurveillance

Normal small intestine

Transfer of soluble antigens via goblet cells to tolerogenic CD103+ DCs in the lamina propria

Tolerance induction against food allergens

γδ T cells

Normal dermis

Slow constitutive migration of a subset of dermal γδ T cells. Interactions with MHC class II+ APCs in the dermis

Immunosurveillance

Mast cells

Normal dermis

Mast cells are immobile in normal dermis. Extensions of dendrites by perivascular mast cells into venules facilitate capture of IgE

Dermal immunosurveillance and monitoring of molecules in circulation

Group 2 innate lymphoid cells (ILC2s)

Normal dermis

Constitutive migration of ILC2 subsets in the dermis and interactions with dermal mast cells

Immunosurveillance

128

Invariant NKT (iNKT) cells

Normal liver

iNKT cells patrol the liver sinusoids via crawling along the vascular wall

Immunosurveillance

130

Liver B. burgdorferi infection

iNKT cells are attracted to Kupffer cells in a CXCR3‑dependent manner

Initiation of immune responses and pathogen containment

131

Dermal blood vessels

LY6Clow monocytes patrol the vasculature by crawling along the blood vessel wall

Immunosurveillance

132

Inflamed kidney

Upon encounter of mild inflammatory stimuli, patrolling monocytes recruit neutrophils and clear necrotic endothelial cells

Initiation of inflammation and containment of tissue injury

133

Inflamed dermis

Monocytes are recruited to the edge of sterile injury sites around neutrophil clusters

Containment of tissue injury

53

Microglia

Normal and inflamed CNS

Microglia are immobile, but demonstrate dynamic movement of their dendrites. Following sterile injury, the dendrites converge at the site of injury

Immunosurveillance and containment of tissue injury

134,135

Kupffer cells

Liver L. donovani infection

Kupffer cells are present within L. donovani-induced liver granulomas where they present antigen to CD8+ T cells

Initiation of T cell responses

136

Dendritic cells (DCs) Epidermal Langerhans cells (LCs)

Dermal DCs (DDCs)

Gut DCs

Monocytes

124,125 126 77,127 128,129

Macrophages

APCs, antigen-presenting cells; B. burgdorferi, Borrelia burgdorferi; CNS, central nervous system; CXCR3, CXC-chemokine receptor 3; dSEARCH, dendrite surveillance extension and retraction cycling habitude; iNKT, invariant natural killer T; L. donovani, Leishmania donovani; L. major, Leishmania major; LY6C, lymphocyte antigen 6C.

Pseudopodia Dynamic extensions of the cytosol and cortex that cyclically protrude from the cell body. The term is now mostly restricted to extensions, such as filopodia, that are driven by protrusive rather than contractile forces.

Migratory modes of leukocytes in the interstitium As for most eukaryotic cell motility, forces that are generated and transmitted by the actomyosin cytoskeleton drive the migration of leukocytes9. Different cell types can adopt distinct modes of migration, which are commonly categorized on the basis of the structure and dynamics of the leading edge and the under lying cytoskeletal organization10. Leukocytes migrate as individual cells, rather than collectively as a cohesive unit, and in the complex three-dimensional environment of the interstitium, they use a migration mode that is classically termed ‘amoeboid’ (REF. 11) (BOX 1).

Amoeboid migration denotes a loosely defined set of related motility techniques that have various methods of execution11,12 (BOX 1). The intracellular segregation of the small GTPases RHOA, RAC and CDC42 gives rise to the polarization of leukocytes during migration (FIG. 1). The type of protrusions (known as pseudopodia) that are formed at the leading edge of a cell, and ultimately the mode of migration the cell uses, is determined by an intricate equilibrium between the protrusiveness of the actin network, the contractility of the actomyosin machinery and the adhesive propensity of its cortex 11,13 (FIG. 1).

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REVIEWS Box 1 | Diversity in motion: the different modes of single cell migration Amoeboid migration is characterized by a rounded shape and a dynamic leading edge, and has evolved into a loosely defined concept that encompasses various subtypes112,113. Pseudopodial or gliding migration most closely resembles that of amoeba, and features a stable uropod at the rear of the cell, with the cell undergoing relatively minor overall shape changes. The small GTPases RAC and CDC42 polarize to the leading edge where they promote ARP2/3 complex-driven actin polymerization, which extends the plasma membrane into pseudopodia, whereas RHOA-driven myosin contractility is concentrated at the mid-body of the cell47. The rear of the cell is rich in myosin motors, septins, and ezrin, radixin and moesin (ERM) proteins, which are known to inhibit protrusion formation and thus contribute to the directional maintenance of cell polarity during migration1,114–116. The cell rear can be dragged along by protrusive and non-adhesive forces at the leading edge17. To date, all leukocyte migration observed in vivo is of the pseudopodial amoeboid mode, although a blebbing type of migration has also been stipulated. Blebbing migration involves the propulsion of the cell body by contractions that are generated by the actomyosin cortex at the rear of the cell, and are transmitted by cytosolic hydrostatic pressure to the leading edge, where expansive forces result in dynamic spherical herniations called blebs12. An over-contractile actomyosin cortex assembles de novo during the expansion phase of the bleb, facilitating its arrest117,118. The concomitant contraction of the cell rear results in a net forward displacement of the cell16,119. Blebbing migration is mainly adhesion-independent, and has been observed in vivo in zebrafish and Drosophila melanogaster primordial germ cells, but it remains to be unequivocally confirmed in mammalian leukocytes119,120.

Molecular clutch As actin filaments undergo retrograde flow, proteins that link actin to integrins transmit cytoskeleton-mediated forces to the extracellular matrix (ECM), thereby enabling forward traction of cells in adhesion-dependent migration. These linker proteins form part of a molecular clutch mechanism, as they allow for the engagement and disengagement of the actin cortex from the ECM, and thus govern the transience of the adhesiveness of cells to their substrate.

Displacement Distance (measured in μm) between cell location at the start and end of the observation.

Pearl-chain nuclei The transient ‘pearls on a string’ rearrangement of the segmented nuclei of neutrophils that are migrating through a confined space.

Perivascular macrophages A subset of macrophages that localizes in close proximity to post-capillary venules in peripheral organs, including the skin, muscles and central nervous system. These cells are involved in leukocyte recruitment to inflamed tissues.

In principle, interstitial migration of leukocytes can be adhesion-dependent and adhesion-independent. In adhesion-dependent migration, nascent focal adhesions in cell protrusions anchor the leukocyte cortex to the extracellular matrix (ECM), notably via integrins and molecular clutch mechanisms14,15. During such adhesion-dependent migration, in which actin polymerization-driven protrusions propel the leading edge, the retrograde movement of anchored F‑actin pulls on the ECM, resulting in a forward displacement of the cell16,17. Actomyosin-mediated retraction of the cell rear facilitates forward motion, but is not essential18. In adhesion-independent migration, leading edge protrusions intercalate with the porous lattice of the ECM, where lateral expansive forces — either protrusive or hydrostatic — firmly wedge the leading edge of the cell in place, in a process likened to the ‘chimneying’ of climbers12,17,19 (FIG. 1). Further expansion of the leading edge and an elastic linkage to the rear are then transformed into a concerted forward translocation of the cell body 16,17. Leukocytes, including dendritic cells, neutrophils and lymphocytes, migrating in vivo use a predominantly actin-protrusive, integrin-independent migration mode17 (FIG. 1). However, CD4+ effector T cells were recently shown to migrate in an integrin-dependent manner during inflammation20, challenging a generalized ‘one size fits all’ model of integrin-independent leukocyte migration. In addition, dendritic cells can migrate along adhesive and non-adhesive surfaces in vitro by switching between integrin-dependent and integrin-independent mechanisms, without an appreciable decrease in migratory velocity or directionality 16. Furthermore, migration through dense environments requires actomyosin contractions at the trailing edge to overcome the resistance posed by the rigid nucleus,

although myosin activity is dispensable in matrices with low densities17,21. Migration in dense ECM environments is also supported in neutrophils by their ability to form pearl-chain nuclei, which enables their migration through smaller pore sizes than leukocytes that have ovoid nuclei, such as T cells or tumour cells21. Overall, leukocytes can rapidly adapt their cell shape and migratory machinery to the varying conditions of the microenvironment, which enables them to effectively traverse interstitial spaces at high speed. We discuss below the individual steps of the journey of a leukocyte from the intravascular into the extravascular space and into the site of tissue injury, infection or cancer.

Perivascular cells in neutrophil guidance Neutrophils are essential for the clearance of tissue debris and pathogens, such as bacteria and parasites22,23. Owing to these vital activities — as well as their abundance in the circulation and bone marrow — neutrophils are perhaps the most well-studied leukocytes in terms of migratory activity. As for other blood-borne cells, extravasation of neutrophils into the tissues starts with adherence to the inflamed endothelium that lines post-capillary venules, followed by TEM. These processes have been reviewed extensively elsewhere1,2,24 and are not considered further in this Review. Instead, we focus here on the first steps of interstitial migration — namely, abluminal crawling and crossing of the basement membrane — and discuss the increasing evidence suggesting that cells in and around the vessel wall modulate neutrophil entry into the extravascular space1. The concept of a multicellular complex governing microvascular function is well established in the context of the blood–brain barrier, where endothelial cells, pericytes, glial cells, neuronal processes and ECM components form the neurovascular unit (NVU)25,26. The intricate interplay between neuronal and vascular elements at the NVU regulates blood flow and the exchange of molecules and cells between the circulation and central nervous system (CNS). By analogy, we have termed the cellular and structural constituents that are involved in leukocyte extravasation within post-capillary venules in peripheral organs the perivascular extravasation unit (PVEU) (FIG. 2). The PVEU is comprised of endothelial cells, pericytes, perivascular macrophages, mast cells and the basement membrane. Interactions between leukocytes and the PVEU are crucial for diapedesis, and may also affect their subsequent activities in the interstitium. Basement membrane and pericytes. Post-capillary venules are ensheathed by the basement membrane, which is a complex meshwork of intertwined polymers and molecules consisting mainly of collagen IV, laminins, nidogens and sulphated proteoglycans27,28. The basement membrane is important for the integrity of the blood vessel wall, and forms a barrier for egressing leukocytes. Embedded in the basement membrane reside venular pericytes, which are elongated cells that are negative for the chondroitin sulphate proteoglycan NG2 and that vary in density and shape depending on the vessel type

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REVIEWS Uropod –

CD44

Contractile force

F-actin

ERM

ECM

Actomyosin

Hydrostatic pressure propagation

Myosin II Clu tch

Integrin

β

Expansive force

Intercalation of confined region

α

MTOC

–

Direction of migration

RHOA

+

Nucleus

Septin

RHOA-mediated contractility RAC- and CDC42mediated protrusiveness

Plasma membrane + +

Filament sliding RHOA

Mid-body F-actin

Pseudopodium Integrin

ERM CDC42

+ –-

Clu tch

β α

–

Cell cortex

Clutch ARP2/3

Nucleus

+

RAC

Myosin II

+

RAC

–

Septins

ARP2/3

ECM

F-actin

Plasma membrane

+

Plasma membrane

Figure 1 | Polarization of cytoskeletal dynamics governs leukocyte shape and migration. A schematic representation of a polarized leukocyte migrating directionally in a three-dimensional environment using a pseudopodial amoeboid mode. A balance of expansive and contractile forces regulates the shape of the leukocyte. Nature Reviews Contractile forces are localized at the rear and mid-body of the cell, whereas expansive forces at the leading| Immunology edge give rise to protrusions. In the uropod, actomyosin contractility, driven by the small GTPase RHOA, helps to propel the cell body forward via cytosolic flow, and can disengage molecular clutch mechanisms that link the cell cortex to the extracellular matrix (ECM) via integrins. Polarization of CD44 to the uropod recruits ezrin, radixin and moesin (ERM) proteins that strengthen cortical integrity, ensuring that protrusion formation is not favoured at the rear of the cell. In the mid-body, actomyosin contractility can distort the shape of the nucleus in order for the cell to traverse restrictive pore sizes of the ECM. At the leading edge, pseudopodia intercalate the porous network of the ECM. The small GTPases RAC and CDC42 activate the ARP2/3 complex, which drives the polymerization of branched actin networks. The actin filaments are oriented such that the addition of monomers generates protrusive forces that extend the plasma membrane. Hydrostatic pressure generated by distal actomyosin contractility can extend the leading edge of cells into blebs, however, the principle of blebbing migration remains to be proven in leukocytes. In confined environments, pseudopodia can generate enough friction to sustain forward propulsion of the cell body, via intercalation of the ECM. Nascent adhesion sites in the pseudopodia, mainly integrin-based, can facilitate leukocyte motility, but are dispensable. MTOC, microtubule-organizing centre.

Uropod A stable, thin, elongated and contractile posterior protrusion that gives rise to the characteristic ‘hand mirror’ shape of polarized migrating cells.

and the vascular bed29,30 (FIG. 2). The venular basement membrane contains regions that have a low density of matrix proteins — termed low expression regions (LERs)31,32 — which localize to the gaps between pericytes. Intravital imaging studies in the inflamed cremaster muscle vasculature demonstrated that, after TEM, neutrophils enter the subendothelial space and then migrate along pericyte processes towards LERs in a process that depends on intercellular adhesion molecule 1 (ICAM1), lymphocyte function-associated antigen 1 (LFA1; also known as integrin-αL) and MAC1 (also known as integrin-αM and CD11b)33. Neutrophils then extend filopodia through LERs to probe the extravascular tissue compartment. Completion of transmigration

is preceded by a marked elongation of the uropod, which remains transiently tethered on the basement membrane via LFA1‑mediated adhesion, as the neutrophil front migrates into the interstitial space34. Perivascular macrophages and mast cells. The existence of dendritic-shaped macrophages in close proximity to the blood vessel wall is well appreciated (FIG. 2). These cells, termed adventitial or perivascular macrophages, have been described, for example, in the skin35, skeletal muscles36 and CNS37,38. Perivascular macrophages discontinuously cover post-capillary venules, where they reside outside the basement membrane. Until recently, little was known about the function of these cells, although they have been



REVIEWS a

Low expression region

Perivascular macrophage

Post-capillary venule

Endothelial cell

Circulating Tethering

Rolling Sticking Transendothelial migration Paracellular

Neutrophil

NG2– pericyte Basement membrane

b

Mast cell

Transcellular

Abluminally crawling neutrophil

PSGL1

LFA1 (low-affinity conformation)

LFA1 (high-affinity conformation)

Selectins

ICAM1 and ICAM2

Chemokine displayed on cell surface

Gα-coupled receptor

Chemokines

Interstitial space

c

Perivascular extravasation unit Basement membrane Low expression region

NG2– pericyte Endothelial cell

Pericyte


Figure 2 | The perivascular extravasation unit assists in neutrophil emigration. a | Schematic representation of the perivascular extravasation unit (PVEU), consisting of endothelial cells, the basement membrane, pericytes, perivascular Nature Reviews | Immunology macrophages and mast cells. After firm adherence in post-capillary venules, neutrophils transmigrate through the endothelial cell monolayer. Following transendothelial migration, neutrophils crawl along pericytes that are negative for the chondroitin sulphate proteoglycan NG2 (NG2– pericyte). This abluminal crawling is dependent on intercellular adhesion molecule 1 (ICAM1), leukocyte function-associated antigen 1 (LFA1) and MAC1. Abluminally crawling neutrophils extravasate from regions of low extracellular matrix protein densities in the basement membrane (low expression regions). Perivascular macrophages may assist in the formation of the extravasation ‘hot spots’ due to their ability to produce large quantities of chemokines. Mast cells present in close proximity to the blood vessel wall may also respond to danger signals with the release of chemoattractive factors. b | High magnification schematic representing abluminal crawling of extravasating neutrophils along NG2− pericytes in an ICAM1-, LFA1- and MAC1‑dependent manner. c | Cropped confocal image of dermal whole mount from a DPE-GFP mouse (which expresses green fluorescent protein (GFP) under the control of the Cd4 enhancers and promoter). The association of perivascular macrophages (green; marked by GFP expression) with pericytes (red; stained with α-smooth muscle actin-specific antibody) and endothelial cells (blue; stained with CD31‑specific antibody) is shown. PSGL1, P-selectin glycoprotein ligand 1.

implicated in antigen presentation to T cells traversing the blood–brain barrier in inflammatory CNS diseases37,38. Using a mouse strain in which perivascular macrophages were selectively marked by transgenic expression of green fluorescent protein under control of the Cd4 enhancers and promoter, perivascular macrophages were recently

found to regulate firm adhesion and transmigration of neutrophils into skin infected with the bacterial pathogen Staphylococcus aureus 39. As visualized by intravital multi-photon microscopy, neutrophil egress preferentially occurred in close proximity to perivascular macrophages. Consistently, perivascular macrophages — but not other

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REVIEWS dermis-resident leukocytes — rapidly upregulated mRNA encoding CXC-chemokine ligand 1 (CXCL1) and CXCL2 following infection with S. aureus 39. The discontinuous coverage of venules suggests that chemokines secreted by perivascular macrophages into the perivascular space may, after transendothelial transport40, be presented in circumscribed areas that contain perivascular macrophages. This could help to explain the occurrence of neutrophil extravasation hot spots along post-capillary venules41. In addition, perivascular macrophage-derived chemoattractants may guide leukocyte migration immediately following egress from the blood vessel wall. Future studies are needed to dissect the crosstalk between perivascular macrophages, pericytes and endothelial cells, and emigrating leukocyte subsets during different inflammatory conditions. Mast cells, which typically also reside in a perivascular location (FIG. 2), have been implicated in the sensing of microbial invasion via their expression of pattern recognition receptors, such as Toll-like receptors42. Upon activation, mast cells can instantly release their granule contents into the surrounding tissue. Pre-stored mediators include the pro-inflammatory cytokines tumour necrosis factor (TNF) and interleukin‑6 (IL‑6), and chemokines, such as CXCL1 and CXCL2, as well as several vasoactive factors, including histamine42. Moreover, mast cells can synthesize de novo chemoattractants and vasoactive substances, including chemokines and lipid mediators (such as prostaglandins, leukotrienes and thromboxanes), which may then contribute to the prolonged attraction of inflammatory cells42.

Neutrophil extracellular traps (NETs). Webs of chromatin fibres that trap and kill microorganisms. Chromatin from the nuclei of neutrophils is extruded to form these extracellular nets, which also contain proteases from the azurophil granules of neutrophils.

Priming of neutrophil effector functions by the PVEU. Besides regulating diapedesis, signals derived from the cellular and non-cellular components of the PVEU have the potential to induce the effector functions of egressing neutrophils that are required for their subsequent interstitial journey and for effective killing of pathogens or clearance of tissue debris43. Indeed, transmigrated neutrophils display an activated phenotype that is characterized by a polarized cell shape, and increased expression of integrins and proteases (reviewed in REF. 43). For example, transmigration of neutrophils through IL‑1β‑stimulated cremaster muscle venules results in PECAM1 (platelet endothelial cell adhesion molecule 1)‑mediated upregulation of α6β1 integrin expression, which facilitates neutrophil movement across the basement membrane44. In addition, signalling via CXCR1 mediates the release of α‑defensins and stimulates a respiratory burst in neutrophils, which is important for bacterial killing 45,46. Given that perivascular macrophages are the major source of CXCR1 ligands, at least during early bacterial inflammation39, it is possible that the PVEU is not only crucial for guiding neutrophils into the extravascular space, but also for their subsequent interstitial locomotion and for priming them for encounter with microorganisms or damaged cells. Together, intravital imaging studies have shed light on the events that immediately follow neutrophil TEM in vivo, and have implicated perivascular cells in the regulation of this process.

Interstitial neutrophil migration Following entry into the interstitial space, neutrophils encounter a large variety of pro-migratory and antimigratory factors, including chemokines, peptides derived from ECM fibre cleavage, molecules containing damage-associated molecular patterns (DAMPs) released from necrotic cells (for example, purinergic nucleotides), formylated peptides (such as N-formylmethionyl-leucyl-phenylalanine (fMLP)) derived from pathogens or dying cells, lipid mediators and complement factors27,47. Intravital microscopy studies in rodent and zebrafish models of inflammation have greatly contributed to our understanding of the dynamics and spatial organization of directed neutrophil migration towards sites of tissue injury or pathogen deposition (reviewed in REFS 23,48,49). These studies have revealed several important concepts: first, neutrophils communicate with each other through the release of mediators that amplify their own recruitment; second, hierarchies of chemoattractants are established within tissues that guide neutrophils over great distances; third, neutrophils use certain scaffolds within tissues that constitute routes of preferential migration; and finally, neutrophils are equipped with a sensory machinery that enables the interpretation of established chemoattractant gradients and facilitates the rapid adaptation of migratory modes. Neutrophil attraction towards sites of damage, and swarming behaviour. Intravital real-time imaging of lymph nodes, skin, liver, lungs and the CNS has been used to track neutrophil behaviour in the interstitium in mouse models of sterile injury and infection50–58. These studies have uncovered the remarkably coordinated kinetics of migrating neutrophils within intact organs. Following intradermal infection with Leishmania major parasites, neutrophils were found to exit blood vessels preferentially at the side facing the infectious inoculum52. Assuming a polarized cell shape, they then migrated with high directionality towards the site of parasite deposition52. Similar directed migration was observed towards sites of sterile injury or after intradermal injection of S. aureus 51,53,56. Neutrophils typically navigate through the interstitium at an average speed of ~10 μm per minute, with peak velocities reaching 30 μm per minute. Once at the focus, they slow down, which is probably associated with the initiation of effector functions, such as phagocytosis and the release of neutrophil extracellular traps (NETs)59. Multiple neutrophils (up to several hundred) then cluster around the focus until the recruitment of new cells ceases. Neutrophil clusters have two main migration patterns. In stable clusters, neutrophils considerably decrease their motility and enter a state of migratory arrest, which is observed following tissue damage by physical trauma, for example. Consequently, they form a seal around the site of damage to contain the injury 51–53,57. Alternatively, sustained active migration of neutrophils within clusters results in the ‘swarming’ behaviour that is commonly seen at infectious foci50. Although sustained migration has also been observed in stable clusters, swarming may lead to the transient assembly and disassembly of these aggregates50.



REVIEWS Step 1: scouting NG2– pericyte

Chemokine


Step 2: amplification

Endothelial cell

Basement membrane

Neutrophil NG2+ pericyte

Step 3: stabilization

Monocyte

DAMPs and/or PAMPs

Necrotic neutrophils

Injury site

Macrophage migration inhibitory factor

Capillary or arteriole

ECM devoid region

ECM Draining lymphatics

Figure 3 | Three-step cascade guides neutrophils to the site of sterile injury in the skin. A schematic representation of the individual phases of neutrophil attraction towards tissue injury sites.Nature Step 1:Reviews following an insult, | Immunology scarce ‘scouting’ neutrophils are attracted towards the injury site. Step 2: activated ‘scouts’ then amplify the response by producing leukotriene B4 (LTB4), which enables additional neutrophil recruitment from more distal regions. Neutrophils transmigrating from the vasculature can also be guided to the site of injury by NG2+ pericytes, which are present around capillaries and arterioles and express macrophage migration inhibitory factor. Neutrophil accumulation at the injury site modifies the extracellular matrix (ECM), resulting in an ECM-free region in the centre of the growing neutrophil cluster. Step 3: large numbers of neutrophils form stabilized clusters thereby containing tissue injury. At this stage, further attraction of neutrophils ceases. Monocytes are present in the periphery of the clusters. Some neutrophils migrate away from clusters to the draining lymphatics, whereas others may also enter the vasculature and then travel to distant organs. Tracks (arrows and lighter coloured cells) for a few neutrophils are shown to highlight the migration pattern of individual cells in the interstitial space. DAMPs, damage-associated molecular patterns; PAMPs, pathogen-associated molecular patterns.

Chemotaxis The process of directional cell migration towards soluble, freely-diffusing gradients of chemoattractants.

Chemokinesis A phenomenon used to describe cells responding to soluble pro-migratory cues with non-directional migration. Soluble, short-lived chemoattractive or chemorepulsive gradients can be rapidly adjusted to enhance or suppress leukocyte migration, respectively. However, the extent to which soluble chemotactic gradients are established within tissues is currently unclear.

A detailed characterization of neutrophil migration dynamics at the population level has revealed a surprising choreography underlying this process. Specifically, a sequential, multistep attraction cascade coordinates cluster formation, and includes scouting, amplification and stabilization phases (FIG. 3). Initially, a small number of individual neutrophils that are close to the damage site — termed scouts or pioneers — migrate towards the focus in a pertussis toxin-sensitive manner, indicating the involvement of Gαi-coupled receptor signalling, to form a small cluster 51,53. Recent evidence suggests that neutro phils are occasionally present even in non-inflamed peripheral organs, such as the skin, where they may act as the first responders to tissue damage51. Early-arriving neutrophils may undergo cell death53, which precedes the second phase of neutrophil attraction: amplification. During amplification, large numbers of additional neutrophils in the interstitial space are attracted from further away (200–300 μm)51,53. This phase depends on neutrophil-produced leukotriene B4 (LTB4), which acts as a paracrine communication signal between neutrophils53 (FIG. 4a). Previous in vitro data demonstrated that LTB4 amplifies neutrophil polarization and chemotaxis towards fMLP, thereby relaying information between individual cells over long distances60. Another molecule implicated in the amplification phase is NAD, which is released from dying cells and is metabolized to the second messenger molecule cyclic ADP ribose (cADPR)

via binding to the ADP-ribosyl ectoenzymes CD38 and CD157 (REF. 61). cADPR is required for the migration of neutrophils to certain chemoattractants, such as fMLP62. Blocking of cADPR signalling in neutrophils abolished amplification, and led to the premature dissolution of nascent clusters generated by neutrophil scouts51. During the final phase, cluster stabilization, neutrophils remodel the ECM, which results in the disappearance of collagen fibres from the cluster core53. Integrins and G proteincoupled receptors, including CXCR2, N‑formyl peptide receptor 2 (FPR2) and LTB4 receptor, mediate the migration and/or retention of neutrophils in the collagen-free zone53. The cessation of additional neutrophil recruitment coincides with the appearance of monocytes at the edge of the clusters53 (FIG. 3). Taken together, an intricate multistep ‘signal–relay’ system ensures that neutrophils arrive rapidly, and in appropriate numbers, at sites of tissue damage (FIG. 3). Dying cells release DAMPs — such as purinergic nucleotides and formylated peptides — which initiate neutrophil attraction. Lipid mediators and chemokines then amplify the response to attract large numbers of additional cells, before recruitment ceases. Decision making during interstitial neutrophil migra tion. It has long been postulated that migrating leuko cytes follow concentration gradients (from low to high) of soluble (via chemotaxis or chemokinesis) or

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REVIEWS a Skin injury model Chemokine gradient Neutrophil Endothelial cell

NG2– pericyte Perivascular macrophage

NG2+ pericyte

LTB4 tissue gradient

Immobilized MIF Capillary or arteriole

Necrotic core

Injury site

Chemokines

PAMPs and DAMPs

Lipid mediators

LTB4 microgradient

Figure 4 | Context-dependent mechanisms of neutrophil attraction to injury sites. a | Model depicting the cascades of chemoattractive gradients, which mediate the centripetal motion of neutrophils towards injury sites via signal relay. Evidence was compiled from several studies in various models51,53,57,60,63,66 to indicate the biochemical basis underlying the proposed multistep attraction cascade. The formation of distinct gradients includes damage-associated molecular patterns (DAMPs), for example, purinergic nucleotides or formylated peptides (such as fMLP), that are released directly at the injury site, chemokines produced by tissue-resident cells or attracted leukocytes, and lipid mediators, such as leukotriene B4 (LTB4), released by neutrophils themselves. Interpretation of these gradients by intracellular signalling molecules allows neutrophil guidance towards the damage focus. b | The production of hydrogen peroxide (H2O2) by epithelial cells establishes a chemotactic gradient for neutrophils during the wound-healing response in zebrafish63. c | The schematic depicts a mechanism for the recruitment of neutrophils to the site of sterile hepatic injury57. Neutrophils initially migrate along intravascular chemokine gradients in the liver sinusoids, followed by an abrupt switch to fMLP gradient sensing close to the necrotic focus. MIF, macrophage migration inhibitory factor; PAMP, pathogen-associated molecular pattern.

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Haptotaxis The process of directed cell migration along immobilized chemoattractant gradients, which may be adhesiondependent or adhesionindependent. Many chemoattractants can be immobilized by binding to heparan sulphates presented on cell surfaces or extracellular matrix fibres. These gradients are generally long lived and are crucial for guiding leukocytes in the interstitial space.

Haptokinesis Random cell motility along two-dimensional surfaces or within three-dimensional spaces guided by immobilized chemoattractants. In two-dimensional haptokinesis, migration relies on firm substrate adherence of the migrating leukocyte, which is mostly mediated by integrins. During three-dimensional haptokinesis, leukocyte motility in the extracellular matrix network is mostly independent of adhesion. The spatial confinement of the migrating leukocyte is sufficient to provide mechanical or weakly-adhesive anchorage to assist in migration.

Chemorepulsion The migration of leukocytes away from certain mediators.

matrix-bound (via haptotaxis or haptokinesis) chemo attractants within tissues. Gradient sensing could explain how leukocytes reorientate their migration machinery within asymmetric concentrations of mediators, resulting in forward movement. However, only recently has experimental evidence provided confirmation of this concept. Imaging studies in a zebrafish larvae woundhealing model revealed the rapid establishment of a hydrogen peroxide (H2O2) gradient at the wound margin that extended up to 200 μm into the surrounding tissue63 (FIG. 4b). Sensing of this gradient via the SRC family tyrosine kinase LYN increased the migratory directionality of neutrophils towards the wound63,64. Notably, H2O2 was dispensable for leukocyte recruitment during bacterial infection in zebrafish65, indicating that there are distinct requirements for neutrophil attraction towards sterile and non-sterile foci. Another study in zebrafish uncovered gradients of CXCL8, the homologue of human IL‑8, at sites of bacterial infection66. This CXCL8 gradient increased the speed of directional neutrophil migration towards the site of infection, and restricted the motility of cells that were close to the focus. In mice, an intravascular gradient of CXCL2 was shown around areas of chemically-induced, sterile liver necrosis, and this supported the CXCR2‑dependent attraction of neutro phils towards the injury zone57 (FIG. 4c). The concept of chemokine gradient-mediated leukocyte guidance has also been recently demonstrated for dendritic cells in the dermis, where a CC-chemokine ligand 21 (CCL21) gradient promoted their haptotactic migration towards lymphatic vessels67. Another possible means of signal diversification by multiple chemoattractants is through spatially and temporally segregated expression. Indeed, in a liver injury model57, the CXCL2 gradient ended abruptly at ~150 μm from the necrotic core, and was then replaced by a gradient of fMLP, which was probably released from dying cells. At this point, neutrophils switched from CXCR2‑guided to FPR1‑guided attraction towards the damage focus (FIG. 4c). Chemokines may also be produced in sequential waves, leading to the graded attraction of leukocyte subsets. During immune complex-mediated arthritis in mice, a temporal ‘lipid–cytokine–chemokine’ cascade regulates neutrophil influx into the joints 68. Initially, small numbers of neutrophils are recruited and activated by LTB4, which results in their production of IL‑1β. This leads to the release of CCR1 ligands by the synovial tissue, followed by the production of CXCR2 ligands by the neutrophils themselves. In summary, the temporally and spatially contained deposition of chemotactic gradients exerts control over the attraction of leukocytes to sites of damage. Besides using chemoattractive gradients within tissues, it is possible that leukocytes use preformed locomotion pathways, for example, those formed by cells or ECM fibres, to determine cell directionality within tissues47. For example, naive T cells in the lymph node physically contact the fibroblastic reticular cell (FRC) network, and they use these structures as guidance cues, possibly via sampling for immobilized chemokines,

such as CCL21 (REFS 69,70) . Such a deterministic migration concept, whereby cells follow preformed anatomical scaffolds, has also recently been proposed for neutrophils migrating in inflamed skin 54 (FIG. 4a). Intravital multi-photon microscopy revealed that following extravasation, neutrophils contacted pericytes that line capillaries and small arterioles, resulting in adhesive interactions mediated by ICAM1 and haptotactic macrophage migration inhibitory factor (MIF). Neutrophils that contacted pericytes migrated faster and more directionally towards a sterile damage focus than non-interacting neutrophils54. Taken together, it is possible that cellular structures can instruct leukocytes with distinct motility programmes that promote enhanced navigation in the interstitial space. Finally, how do leukocytes make migratory choices during the simultaneous encounter of competing chemo attractive gradients? In vitro studies have shown that neutrophils can integrate multiple chemotactic signals and then prioritize their migratory response. Specifically, ‘end target’ chemoattractants — such as the N‑formyl peptide fMLP or the complement protein C5a — override signals from the chemokines that are implicated in homing, for example, CXCL8 or LTB4 (known as intermediate chemoattractants)71–73. Studies by Kubes and colleagues73 demonstrated that these two classes of chemoattractants induce differential signalling pathways in neutrophils: p38 mitogen-activated protein kinases (MAPKs) mediate the effects of fMLP and C5a, whereas the phosphatidylinositol 3‑kinase (PI3K) γ‑subunit is responsible for the effects of CXCL8 and LTB4. In addition, phosphatase and tensin homologue (PTEN) suppresses PI3K activity in neutrophils migrating within opposing chemokine gradients, thereby preventing the ‘distraction’ of neutrophils by signals of subordinate importance74. The precise function of these signalling cascades during interstitial migration in situ requires further investigation. Chemorepulsion and reverse transmigration of neutro phils. Under certain circumstances, neutrophils have also been observed to migrate away from sites of infection or damage. The biochemical basis for this phenomenon is poorly understood, but parallels have been drawn with cellular migration in the CNS, where bidirectional cues regulate axonal outgrowth75. Indeed, several factors that facilitate the repulsive guidance of axons, including slit, netrins and repulsive guidance molecule A, have also been implicated in repelling neutrophils75,76. Chemorepulsion could have a negative regulatory role during inflammation, but it may also contribute to the phenomenon of reverse transendothelial migration (rTEM); that is, the movement of neutrophils from the interstitium into the blood or lymphatic vessels (FIG. 3). For example, pathogen-laden neutrophils have been detected in lymphatic vessels during mycobacterial skin infection77, in which they transport pathogens to the draining lymph nodes. rTEM of neutrophils also occurs during ischemia–reperfusion injury and is inhibited by the expression of junctional adhesion molecule C41. Notably, after rTEM, neutrophils exhibit an activated

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REVIEWS ICAM1hi phenotype, and are redirected to distal organs — such as the lungs — where they induce secondary inflammation. In zebrafish, the final stages of woundhealing responses are paralleled by the rTEM of neutrophils, which are then dispersed throughout the animal78. Thus, although the precise mechanisms underlying rTEM remain to be determined, this phenomenon may contribute to the resolution of inflammation. Taken together, we have come a long way in our understanding of the molecular and cellular basis of neutrophil guidance towards foci of tissue damage and infection. Future studies will have to focus on a further dissection of the intracellular machinery that allows leukocytes to interpret migratory cues in vivo.

Interstitial migration of effector T cells Similarly to other leukocytes, the basic principles of amoeboid motility and interactions with the environment also apply to effector T cells. However, distinct migratory behaviours need to be considered, which arise from specific activities of T cells during immune responses4,5,7,79. The induction of effector functions requires contact between T cells and the target cells bearing cognate antigen on their surface. Since target cells may be rare and interspersed with other cells within densely packed inflammatory sites, or may diminish in numbers over the course of an immune response, invading T cell populations have to devise optimized search strategies, which may be less reliant on chemoattractive signals than is the case for neutrophils. Similarly to naive T cells within lymph nodes, effector T cell populations therefore have a ‘scanning’ behaviour within peripheral sites, which ensures that targets are not overlooked. T cell migration at effector and memory sites. TEM of T cells is less well understood compared with that of neutrophils, and is not further considered here. Subsequent to extravasation, effector CD4+ and CD8+ T cells polarize and migrate at high speed (5–10 μm per minute) through the interstitial space7. During their interstitial navigation, they exhibit considerable shape plasticity and occasional short migratory pauses that are characterized by ‘rounding up’, which is similar to the ‘stop–and–go’ behaviour of naive T cells in lymph nodes6. It is currently unclear whether such shape oscillations are the result of a cell-intrinsic migratory programme or are extrinsically imposed, for example, by environmental obstacles79. A key regulatory cue for migrating T cells is provided by T cell receptor (TCR) signalling, which results in a shutdown of migratory activity during encounters with cognate antigen. This TCR-mediated ‘stop signal’ hypothesis was originally proposed on the basis of in vitro studies showing that the encounter of peptide–MHC complexes mediated the migratory arrest of activated T cells that were crawling on immobilized ICAM1 (REF. 80). Intravital multi-photon imaging studies later revealed that T cells migrating within effector sites cease migration following engagement with cells that express cognate antigen81–83, which indicates that the TCR also delivers migratory stop signals in vivo.

Since the main function of cytotoxic T lymphocytes (CTLs) is the directed delivery of cytotoxic mediators to target cells via the lytic synapse, interstitial migration mainly serves to bring CTLs into close contact with their targets. Indeed, as visualized in subcutaneously implanted tumours, a large proportion of T cells physically contact tumour cells in the early phase of tumour rejection81,82 (FIG. 5a). Such interactions can be transient (lasting for minutes) or can last for up to several hours, either without obvious effects on the tumour cells, or resulting in their eventual killing81,82. Studies using cell targets labelled with a fluorescent apoptosis indicator have estimated that the CTL killing rate is approximately one cell every 6 hours84, which is considerably longer than that observed during cell killing in vitro. At later stages of the antitumour response, CTLs regain high motility, which supports the scanning of the tumour bed for residual malignant cells. Maintenance of a highly migratory phenotype seems to depend on continued signals from the TCR, as antigen-nonspecific effector CTLs cease migration within tumours81. At sites of chronic infection, for example, in the brain during Toxoplasma gondii infection, CTLs also contact target cells85,86. Although CTLs transiently arrest in an antigendependent manner near isolated parasites — which are frequently found in granuloma-like structures that contain antigen-presenting cells (APCs) — they seem to ignore cysts formed by T. gondii 86. Consistent with the tumour model, the highest migratory velocity of CTLs during toxoplasmic encephalitis was observed at a stage when the maximum numbers of T cells were present in the brain and coincided with a reduced antigen load85. The behaviour of CD4+ effector T cells has been studied in various models of autoimmunity and infection. For example, during experimental autoimmune encephalomyelitis, antigen-specific CD4+ T cells enter the spinal cord parenchyma, with two-thirds of the cells displaying fast, unrestricted migration, and the rest interacting with target cells in an MHC class II‑dependent manner 87. Subsequent studies using adoptive transfer of T helper 17 cells revealed that T cells also contacted neurons and axons in demyelinating lesions, albeit independently of the TCR88. During L. major infection in the skin, CD4+ effector T cells undergo stable and dynamic interactions with infected APCs, although not all infected cells induced the migratory arrest of T cells89. Remarkably, however, stably interacting T cells produced an interferon‑γ (IFNγ) gradient that extended up to 80 μm beyond the site of antigen presentation, thereby triggering antiparasitic responses in infected bystander cells90. CD4+ effector T cells can also enter granulomas that are established during mycobacterial infection in the liver, where they show rapid (albeit confined) migration, which seems to be imposed by granuloma-resident macrophages91. However, antigen-induced T cell arrest in granulomas was infrequent, which was attributed to the low levels of antigen presentation in these sites92. T cells also produced low levels of effector cytokines under these conditions, thereby indicating that migratory stop signals are closely linked to the induction of effector function.



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Figure 5 | CD8+ effector T cell behaviour in the tumour microenvironment. Schematic representation of cytotoxic T lymphocyte (CTL) behaviour in the tumour microenvironment. a | CTLs enter the tumour bed through blood vessels in the periphery of solid cancers. Upon entering the tumour, T cells contact extracellular matrix (ECM) fibres, which potentially serve as haptotactic migration scaffolds. Migrating CTLs also can be repelled by dense ECM fibre bundles around tumour cell nests. CTLs scan both antigen-presenting cells (APCs) and tumour cells for the presence of cognate antigen. Serial contacts of CD8+ T cells with APCs can lead to their activation as shown by nuclear factor of activated T cells (NFAT) translocation into the nucleus. Regulatory T (TReg) cells potentially shorten the duration of these contacts, contributing to the development of tolerance. Activated T cells interact with cognate antigen-positive tumour cells, resulting in the formation of a lytic synapse. Release of cytoxic mediators into the cleft between a CTL and a tumour cell leads to tumour cell apoptosis. The apoptotic debris is phagocytosed by macrophages present in the tumour microenvironment. Tracks (arrows and lighter coloured cells) for a single CD8+ T cell are shown to highlight the migration pattern in the interstitial space. b | Schematic representation of potential migratory dynamics of CD8+ T cells undergoing Brownian walk movement, or — as recently demonstrated in infected brains — Lévy walk behaviour. Lévy walks facilitate the scanning of a larger tissue area by CD8+ T cells. FASL, FAS ligand; ICAM1, intercellular adhesion molecule 1; LFA1, lymphocyte function-associated antigen 1; TCR, T cell receptor.

The behaviour of memory T cell populations during tissue surveillance has recently been studied in a cutaneous herpes simplex virus infection model. Surprisingly, herpesvirus-specific CD4+ and CD8+ memory T cell populations segregated into the dermis and epidermis, respectively 93. While dermal memory CD4+ T cells continued active migration, CD8+ T cells acquired

b

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a dendritic shape and crawled at low speed between keratinocytes. Furthermore, CD4+ T cells could enter systemic circulation, whereas CD8+ T cells remained in the epidermis, where they could rapidly recognize rare antigen-expressing epithelial cells93,94. The strict separation of CD4+ and CD8+ T cells into dermal and epidermal compartments may be pathogen-specific, as

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REVIEWS after infection with vaccinia virus, virus-specific CD8+ memory T cells were also found to reside within the dermis95. However, their migratory kinetics in the dermis are unknown. Functional impairment of T cells and migratory activity. Recent evidence suggests that T cells also display altered migratory patterns during states of functional impairment, a phenomenon that is mediated, at least in part, by co-stimulatory and co-inhibitory pathways. The idea that these pathways can modulate the locomotion of interstitial T cells stems from initial imaging experiments in lymph nodes, where it was found that cytotoxic T lymphocyte antigen 4 (CTLA4) could override TCR-induced stop signals, resulting in increased motility and decreased stable engagement with dendritic cells96. Subsequent studies revealed that, compared with activated control CD4+ T cells, tolerized islet antigen-specific CD4+ T cells displayed increased migratory speed and reduced dendritic cell contacts in pancreas-draining lymph nodes97. These effects could be reversed by blocking the programmed cell death 1 (PD1)–PD1 ligand (PDL1) pathway, but not by inhibiting CTLA4. Similarly, blocking of PDL1 resulted in the reduced migration of tolerized T cells and prolonged the contacts with dendritic cells in the pancreatic islets97. Conversely, during persistent viral infection, exhausted T cells in the splenic red pulp seemed to be locked in a state of motility paralysis98. Reversal of this paralysis via blockade of the PD1–PDL1 pathway not only resulted in increased locomotion, but also in the recovery of effector function, including IFNγ production. As a consequence, a fatal inflammatory disease was triggered, which suggests that interference with T cell migration may protect the host from potentially deleterious immunopathology during certain infections. In summary, it seems that signals that are delivered through the TCR and co-stimulatory and/or inhibitory pathways, together with the activation state of T cells, are important regulatory cues for interstitial migration. The fact that modulation of these signals can restore — in a context-dependent manner — efficient T cell migration helps to explain some of the therapeutic mechanisms of drugs that are currently in clinical use, including antibodies that block CTLA4, PD1 and PDL1. Patterns and mechanisms of interstitial effector T cell migration. The pattern of T cell populations that migrate within effector sites can be best described as random walk (Brownian motion), which is similar to the behaviour of naive T cells within the lymph nodes6,79 (FIG. 5b). Thus, rather than moving in a certain direction, individual effector T cells meander away from an arbitrary starting point, and this has been taken as evidence for the lack of large-scale soluble chemokine gradients within tissues. In the lymph node, naive T cells follow the FRC network, giving rise to the concept of ‘guided random walks’, whereby cells take random migratory decisions at ECM bifurcation points69.

Circumstantial evidence suggests that effector T cells within subcutaneous tumours81,82, the infected brain85 or the inflamed skin99 also crawl along ECM fibres, which indicates a potential role for ECM receptors in this process. The membrane molecule CD44 — which is a receptor for hyaluronan and recruiter of cortexmembrane linkers, such as ezrin (FIG. 1) — has been shown to control interstitial CTL migration within tumours100. Surprisingly, however, this occurred independently of its function as an ECM receptor, but rather through the stabilization of a polarized cell shape in migrating T cells. By contrast, a recent study demonstrated that interstitial motility of CD4+ effector T cells in the inflamed dermis depended on the fibronectin receptor αV integrin (also known as ITGAV)20. Skin inflammation resulted in marked remodelling of ECM components, with fibronectin deposition along collagen fibres, and T cells moved along these fibres. Administration of blocking antibodies against αV integrin led to the migratory arrest of T cells20, which suggests interference with haptotactic migration. In contrast to the proposed promigratory effects of T cell–ECM contacts described in these studies, in human lung cancers, areas of densely aligned fibres around tumour cell islets restrict the access of CTLs to tumour cells101. Hence, the precise role of the ECM in interstitial T cell migration seems to be context-dependent, and can either enhance or restrict T cell locomotion. Finally, recent evidence suggests that the random search strategies of T cells within tissues can be improved by the adoption of generalized Lévy walks, as shown for CTLs in the brain of mice infected with T. gondii 102 (FIG. 5b). Lévy walks are another form of random walk behaviour that is characterized by occasional ‘migratory runs’ with high net displacement, resulting in enhanced tissue penetration. Indeed, cells migrating by generalized Lévy walks find their targets more efficiently than cells migrating by Brownian walks102. Mechanistically, the chemokine CXCL10 supports Lévy walk behaviour by increasing the average migration speed of CTLs, and thereby shortening the time taken to find target cells. Whether T cells also use similar migration strategies in other organs remains to be determined. Taken together, effector T cells may, at least under certain conditions, use migratory scaffolds for their interstitial locomotion, and devise optimized search strategies for rare target cells in the form of generalized Lévy walks. Interactions of effector T cells and APCs. Effector T cells not only contact infected cells or tumour cells but also APCs, including dendritic cells and macrophages, that are present within the effector site. These interactions may be transient or long-lasting, which raises the interesting question: how does the duration of such encounters affect T cell function? Previous in vitro studies using three-dimensional collagen gels have revealed that brief serial interactions between T cells and neighbouring dendritic cells enable the accumulation and integration of signals over time, subsequently resulting in T cell activation103. Three recent studies have used a fluorescently-encoded sensor of nuclear factor of T cells (NFAT) to investigate the outcome of short-lived



REVIEWS T cell–APC interactions in vivo104–106. Following TCR stimulation, NFAT is rapidly translocated from the cytoplasm to the nucleus, thereby serving as a sensitive activation marker. In experimental autoimmune encephalomyelitis, short-term contacts between extravasated T cells and leptomeningeal APCs resulted in efficient, antigen-dependent effector T cell activation105,106. In a tumour model, unstable serial contacts with APCs also led to the nuclear trans location of NFAT in crawling T cells104 (FIG. 5a). This study further showed that nuclear NFAT export was prolonged even after contact had ceased (‘NFAT memory’), and that during this phase T cells transcribed tolerance-associated (but not effector function-associated) genes. In the presence of regulatory T cells within the tumour bed, CTL– APC contacts were destabilized, resulting in increased NFAT memory. This phenomenon could thereby contribute to tumour tolerance. In summary, T cells that establish serial transient encounters with APCs in the periphery may indeed summate signals, although the functional outcome of such signals depends on the nature of the APC, as well as the microenvironmental context of the interactions.

Concluding remarks and outlook Leukocyte motility in interstitial compartments is an essential part of both innate and adaptive immune responses. From the varying shapes and leading edge dynamics that are adopted at a cellular level, to the intricate swarming and scanning behaviours of leukocytes, interstitial migration is a complex feat. Recent advances in intravital imaging technology have provided us with a remarkable new insight into the mechanisms and kinetics of immune cell locomotion in the interstitial space of various organs, but it is evident that we have merely grazed the surface of the unforeseen levels of control that underpin these migratory processes in vivo. Nonetheless, these advances in our understanding of both the biochemical and biomechanical cues involved have opened up the prospect of promising developments in the burgeoning therapeutic field of immune modulation. 1.

2.

3. 4. 5. 6.

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8.

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Interference with leukocyte migration has already been used to moderate excessive inflammation in the clinic, such as that occurring in the context of autoimmune disease107. Leukocyte motility can be targeted during extravasation or subsequent chemoattraction towards foci of tissue damage. In the targeting of extravasation, drugs such as natalizumab, an antibody specific for α4 integrin (also known as ITGA4), and efalizumab, an antibody specific for CD11a (also known as ITGAL; no longer in clinical use), have been successfully used to treat chronic inflammatory conditions by curbing immune cell recruitment 108. Although very effective in certain autoimmune diseases, a potential side effect of these drugs is the disruption of normal immuno surveillance, particularly in the brain. For the inhibition of chemoattraction, a large number of chemokine receptor and lipid mediator (for example, LTB4) antagonists have made it into Phase I, II and III clinical trials107,109,110. In the case of autoimmunity, most of these trials have shown disappointing results, the reasons for which are not entirely clear. The selective abrogation of subset-specific interstitial motility may constitute a more measured and targeted approach to the treatment of inflammatory disorders108. Interference with the intracellular migration machinery of leukocytes represents a promising option in this regard. RHO family GTPases and downstream effectors govern the cytoskeletal dynamics of migrating leukocytes, and thus are also prime pharmacological targets for the inhibition of immune cell recruitment. Conversely, adoptive immunotherapy by exogenous optimization of the migratory potential of effector cells, notably in the context of tumour-infiltrating CD8+ T cells, is evolving into a promising recourse for treating various cancers111. Exciting times lie ahead, and we are on the cusp of converting new lessons learnt from intravital studies into novel effective treatment options and personalized therapies for a range of diseases that have increasing socioeconomic burden, from immune-mediated inflammatory diseases to cancers.

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Acknowledgements

The authors thank members of the Immune Imaging Program at the Centenary Institute, Australia, for critical reading of the manuscript and helpful discussion. This work was supported by the National Health and Medical Research Council, Australia (grant numbers: 1010680, 1030145, 1030147, 1032670 and 1047041), and the Australian Research Council (grant numbers: DP110104429 and DP120103359). W.W. and M.B. were supported by fellowships from the Cancer Institute New South Wales, Australia. M.B. was supported by Cure Cancer Australia Foundation/Cancer Australia grant number1070498 and an ECR grant from the Sydney Medical School, Australia.

Competing interests statement

The authors declare no competing interests.


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