Immunology

The influx and efflux of cells and antigens to and from the draining lymph nodes largely take place through the subcapsular, cortical and medullary sinus systems. Recent analyses in mice and humans have revealed unexpected diversity in the lymphatic endothelial cells, which form the distinct regions of the sinuses. As a semipermeable barrier, the lymphatic endothelial cells regulate the sorting of lymph-borne antigens to the lymph node parenchyma and can themselves serve as antigen-presenting cells. The leukocytes entering the lymph node via the sinus system and the lymphocytes egressing from the parenchyma migrate through the lymphatic endothelial cell layer. The sinus lymphatic endothelial cells also orchestrate the organogenesis of lymph nodes, and they undergo bidirectional signalling with other sinus-resident cells, such as subcapsular sinus macrophages, to generate a unique lymphatic niche. In this Review, we consider the structural and functional basis of how the lymph node sinus system coordinates immune responses under physiological conditions, and in inflammation and cancer. Recent single-cell studies have revealed a previously unappreciated heterogeneity among endothelial cells that line the lymphatic sinuses of the lymph nodes. In this Review, the authors describe these various lymphatic endothelial cell types and how they support the trafficking of cells and antigens through lymph nodes.

Commerford et al. characterize the morphological and molecular changes that occur
in lymphatic endothelial cells (LECs) in tumor-draining lymph nodes (LNs) in two distinct
tumor models. They show an upregulation of integrin αIIb in LN LECs and find that
integrin αIIb mediates LEC adhesion to fibrinogen.

Summary G‐protein–coupled receptor (GPCR) signaling is essential for the spatiotemporal control of leukocyte dynamics during immune responses. For efficient navigation through mammalian tissues, most leukocyte types express more than one GPCR on their surface and sense a wide range of chemokines and chemoattractants, leading to basic forms of leukocyte movement (chemokinesis, haptokinesis, chemotaxis, haptotaxis, and chemorepulsion). How leukocytes integrate multiple GPCR signals and make directional decisions in lymphoid and inflamed tissues is still subject of intense research. Many of our concepts on GPCR‐controlled leukocyte navigation in the presence of multiple GPCR signals derive from in vitro chemotaxis studies and lower vertebrates. In this review, we refer to these concepts and critically contemplate their relevance for the directional movement of several leukocyte subsets (neutrophils, T cells, and dendritic cells) in the complexity of mouse tissues. We discuss how leukocyte navigation can be regulated at the level of only a single GPCR (surface expression, competitive antagonism, oligomerization, homologous desensitization, and receptor internalization) or multiple GPCRs (synergy, hierarchical and non‐hierarchical competition, sequential signaling, heterologous desensitization, and agonist scavenging). In particular, we will highlight recent advances in understanding GPCR‐controlled leukocyte navigation by intravital microscopy of immune cells in mice. 1 INTRODUCTION Innate and adaptive immune responses depend on the coordination and spatiotemporal control of leukocyte dynamics between and within lymphoid organs and peripheral tissues. A wide spectrum of chemically diverse molecules including small proteins, peptides, lipids, nucleotides, and extracellular matrix fragments can act as chemoattractants, regulating leukocyte motility, migration, chemotaxis, positioning, and cell‐cell interaction. Leukocytes perceive these signals mainly through G‐protein–coupled receptors (GPCRs) that, upon agonist binding, induce intracellular promigratory responses.1, 2 Based on the cell type–specific expression of cell surface GPCR type combinations and intracellular GPCR‐controlling protein networks, each leukocyte subset has evolved specific trafficking paths to fulfill their designated effector functions in the body.3-5 Over the last decades, the analysis of GPCR‐deficient mouse models has advanced our understanding of how specific GPCR signals determine immune cell positioning and compartmentalization in situ. Recent work now also started to shed light on the establishment of chemokine patterns and chemotactic fields that guide leukocytes in mammalian tissues.6, 7 In particular, intravital imaging of control and GPCR‐deficient leukocytes in tissues of live anesthetized mice has brought unprecedented insight into the dynamic migration patterns of immune cell subsets in situ.8, 9 Despite these achievements, it is still largely unclear how leukocytes make directional decisions in lymphoid and inflamed organs where they constantly encounter GPCR signals emanating from multiple sources. The detailed mechanisms of how leukocytes sense, integrate, and prioritize between multiple chemotactic fields in situ and how this influences their physiological trafficking are still largely unknown. Dissecting these molecular events experimentally in mammalian tissues is very challenging and demanding. In particular, we are missing important pieces of information (eg, the exact distribution and concentrations of known chemottractant, GPCR dynamics in leukocytes) and cannot control for unknown chemotactic fields. Due to these current limitations many of the emerging concepts for leukocyte navigation in complex chemoattractant milieus come from studies in controlled in vitro systems2 or lower model organisms.10, 11 However, these concepts from reductionist models may not always be easily translatable to the complex situation in mammalian lymphoid organs and inflamed peripheral tissues. In recent years, several systematic studies in mice have started to disentangle how multiple GPCR signals shape leukocyte migration patterns in the complexity of mammalian tissues. In this review, we will summarize common concepts and mechanisms of GPCR‐controlled leukocyte navigation coming from studies with mouse and human leukocytes. We outline the protein networks that control GPCR signaling and GPCR activity and discuss how these may control leukocyte navigation strategies. In particular, we highlight our current knowledge on leukocyte navigation in complex chemoattractant fields and summarize them for three leukocyte subsets (neutrophils, T cells, and dendritic cells [DC]) with different physiological trafficking paths. For each cell type, we will critically evaluate the relevance of current concepts for leukocyte navigation in mouse tissue. While it is very likely that some navigation strategies are only relevant for certain immune cell types and adapted to specific physiological tissue environments, leukocyte subsets may also share basic strategies for directional decision‐making in lymphoid and inflamed tissues of mammals. 1.1 Concepts and mechanisms of GPCR‐controlled leukocyte navigation Many chemokines and chemoattractants are classical inducers of leukocyte polarity, adhesion, and locomotion. They act through GPCRs on intracellular signaling pathways to establish biochemical asymmetry within the cell, preceding the formation of a polarized cell with an actin‐rich front and a myosin II–rich rear.12, 13 Key molecular pathways involved in establishing functional polarity downstream of GPCRs in leukocytes depend on phosphoinositide 3‐kinases (PI3K) and members of the Rho family of small GTPases.14 Rac isoforms, Cdc42 and RhoA represent major signal hubs that act through several effector molecules to regulate actin polymerization and actomyosin cytoskeleton. The balanced interplay of these components shapes the typical amoeboid migration mode of each leukocyte subset.15, 16 In homogeneous fields of soluble chemokines and chemoattractants, most leukocytes undergo self‐polarization, before increasing their motility, migration speed, and movement in a random and undirected fashion (chemokinesis) (Figure 1A).17 Chemokinesis is considered a migratory pattern that helps leukocytes scan large tissue volumes. We have learnt from intravital microscopy studies in mice that lymphocytes show such continuous undirected (random‐walk) migration in lymphoid organs to enable encounters and interactions with other leukocytes.9 Together with other determining factors (eg, confinement between stromal tissue elements), uniform chemokine signals critically control chemokinesis by increasing the speed of randomly moving lymphocytes in situ.18 Once leukocytes perceive external gradients of soluble chemokines and chemoattractants, they can perform directional movement along the concentration gradients of these diffusing attractants (chemotaxis) (Figure 1B).17 Some leukocyte subsets have also been reported to be repelled from high chemoattractant concentrations (chemorepulsion), leading to their movement against the gradient (fugetaxis).19 However, the underlying molecular mechanisms of this process are still under investigation (Figure 1C).20, 21 Apart from soluble molecular cues, leukocytes also perceive surface‐bound attractants as gradients or as homogeneous fields and undergo movement along these surfaces in a directed (haptotaxis) (Figure 1B) or undirected manner (haptokinesis) (Figure 1A), respectively.16, 22 The aspect of leukocyte navigation along surface‐bound attractants is particularly relevant for chemokines with binding capacities to extracellular glycosaminoglycans (GAGs), which decorate many stromal cell elements in tissues.22, 23 This form of imprinting non‐diffusible chemokine gradients into the tissue has been shown critical for leukocyte trafficking across the blood endothelium and entry into lymphatic vessels.6, 23 The terms “chemotaxis” and “haptotaxis” subsume a spectrum of navigation strategies, which leukocytes can use to follow attractant gradients.24 When perceiving an external attractant gradient, some leukocytes (eg, unstimulated neutrophils in culture dish) undergo a full morphological change from unpolarized shape to the establishment of a front‐back axis, which then leads to directional movement along the gradient. In contrast, other leukocytes already migrate with pre‐established polarity (eg, DCs and neutrophils in 3D), before they steer along the gradient by redirecting their migration path. Alternatively, prepolarized leukocytes may move along a gradient without redirecting their path and start to accelerate the closer they get to the attractant source (eg, neutrophils in response to dying cells). Once at the highest attractant source, leukocytes may also actively decelerate and slow down their migration.24 As evidenced from intravital microscopy of select leukocyte subsets, we know that this spectrum of chemotactic responses also occurs in mouse tissues. While we acknowledge that chemokines and chemoattractants are central for many aspects of these migratory responses, we are missing a detailed understanding about how these signals shape the navigation of leukocytes in situ. Is the cellular movement guided by one or more attractant signal(s)? How do leukocytes navigate in complex gradient fields and sense the right direction? How do leukocytes sense their final destination and know where to stop? Are there inherent differences between leukocyte subsets in their navigation strategies? As many other cell types, leukocytes possess several mechanisms that directly regulate the functionality of GPCRs.13, 25-28 These are mainly controlled by GPCR interactions with three protein families: heterotrimeric (α, β, γ) G‐proteins, G‐protein–coupled receptor kinases (GRKs), and β‐arrestins. Upon stimulation, a GPCR functions as a guanine nucleotide exchange factor that catalyzes the exchange of GTP for GDP on Gα subunits of the heterotrimeric G‐protein. In leukocytes, Gα subunits of the pertussis toxin‐sensitive Gi subfamily play critical roles for the signaling of many chemokine and chemoattractant receptors. The dissociation of Gβγ from Gα‐GTP allows them both to independently activate downstream signaling pathways. Leukocyte chemotactic responses are largely controlled by the released Gβγ, which activates phosphoinositide 3‐kinase (PI3K) and phosphoinositide‐specific phospholipase Cβ (PLC)/inositol‐trisphosphate (IP3)/diacylglycerol effector pathways.29, 30 The termination of GPCR signaling relies on the intrinsic GTPase activity of the Gα subunit that hydrolyzes GTP and reassembles as Gα‐GDP with Gβγ to form an inactive heterotrimer. This Gα GTPase reaction is accelerated by a family of proteins, called “regulators of G‐protein signalling” (RGS proteins) to facilitate the rapid turning off GPCR signaling. Hence, RGS proteins are considered key desensitizers of G‐protein‐signaling pathways.27 In addition, another negative feedback loop evolved to prevent the overstimulation of cells through excessive GPCR stimulation. Following agonist binding and G‐protein activation, GPCRs become rapidly phosphorylated on their cytoplasmic tails and intracellular C‐termini by specific enzymes, usually members of the GRK family.31, 32 The pattern of receptor phosphorylation creates a barcode with high‐affinity binding sites for the recruitment of β‐arrestin proteins.33 As a consequence, the increased binding of β‐arrestin to the receptor sterically hinders the GPCR interaction with G‐proteins. This uncoupling of G‐proteins from the receptor, referred to as “receptor desensitization”, prevents further GPCR activation by repeated agonist stimulation over seconds to minutes.34 β‐arrestins can also control more long‐lasting desensitization of GPCRs over minutes to hours. As they can interact with clathrin‐coated pits, their binding to GPCRs also targets receptors for internalization. Besides, GPCR internalization can also follow other pathways (arrestin independent, clathrin independent) or not occur at all.35 Depending on the intracellular trafficking route, the internalized receptors may be subject to degradation, leading to the downregulation of cell surface GPCRs. Alternatively, targeting internalized GPCR to recycling endosomes and back to the plasma membrane can quickly replenish GPCRs on the cell surface.35 In addition to their important roles in receptor inactivation and internalization, GPCR‐recruited β‐arrestins also act as signaling mediators by directly coupling to numerous signaling molecules.36 At many GPCRs, β‐arrestin‐mediated signaling and classical G‐protein signaling can have distinct biochemical and physiological actions from one another. A relatively new paradigm on GPCR signaling considers the allosteric interaction among the ligand, the GPCR, and the transducers (β‐arrestins and GRKs) and how their contribution influences the bias for G‐protein– or β‐arrestin–mediated signaling.28 Resulting from this paradigm, the concept of biased agonism, ie, that different ligands at the same GPCR are able to activate some signaling pathways while blocking others, is of particular interest for many chemokine receptors with prominent roles in leukocyte navigation.37 In the following, we will point out the most commonly and recently discussed mechanisms, which would allow the control of leukocyte navigation. Given the before‐mentioned regulation mechanisms for GPCR functionality, a “One‐GPCR system” appears already sufficient to control chemokinesis and chemotaxis of leukocytes (Figure 2A‐E). To increase the sensitivity for attractant gradients, leukocytes can upregulate the cell surface expression of GPCRs. Some leukocytes store GPCRs in intracellular vesicles, which can fuse with the plasma membrane under conditions of leukocyte activation.38 While the release of GPCR from vesicular stores occurs within minutes to hours, most leukocytes often respond to external stimuli by changing their cell surface GPCR profile over hours to days. These changes are primarily based on the induction of gene‐transcription networks, which also include GPCR proteins, when leukocytes transition between cell states or undergo differentiation (Figure 2A). Multimerization of GPCRs, including homomerization of the same GPCR type, provides another level of GPCR regulation.25 A recent study highlighted the functional importance of GPCR oligomerization in promoting leukocyte chemotaxis (Figure 2B). By lowering cellular cholesterol, inflammatory stimuli enabled GPCR oligomers that acted as scaffolds for distinct promigratory signaling pathways to foster efficient leukocyte migration.39 However, GPCR multimerization has also been reported to dampen GPCR responsiveness.25 Several other mechanisms can attenuate the sensitivity of leukocytes for chemokines and chemoattractants. One involves the presence of a receptor antagonist that binds to the same GPCR as the gradient‐forming attractant agonist, but exerts blocking function for promigratory signaling (Figure 2C). More cell‐intrinsic mechanisms include the downregulation of GPCR expression by lowering GPCR mRNA stability40 or enhancing GPCR degradation.41 As discussed above, agonist‐induced receptor desensitization also attenuates the signaling responsiveness of GPCRs. Upon homologous desensitization, when GRKs phosphorylate agonist‐specific GPCRs, the further recruitment of β‐arrestins can lead to G‐protein uncoupling and internalization of GPCRs (Figure 2D). This process explains why many leukocyte chemotaxis responses toward single attractants are dose dependent and follow a bell‐shaped curve, with higher concentrations resulting in stasis.42 Homologous desensitization may be particularly relevant when leukocytes sense high attractant concentration in situ, eg, at the center of an attractant source.43 Under such conditions, the majority of cell surface GPCRs is likely to undergo desensitization, internalization, and degradation, which may then result in cellular unresponsiveness to continuous or repeated stimulation through the same attractant. As a strategy to remain responsive to subsequent stimulation in gradients, some leukocytes internalize GPCRs, deploy its ligand cargo into lysosomes, and quickly recycle unloaded GPCRs back to the plasma membrane.44 Thus, by eliminating chemotactic factors from the extracellular environments, classic GPCRs can acquire additional scavenging function. This “consumption” of external attractant may also impact the chemotactic milieu by reducing the strength of attractant cues.44 Instead of only binding one agonist, many “One‐GPCR systems” can bind and respond to two or more chemoattractants and activate downstream signaling (Figure 2E). Sensing these distinct attractants through the same GPCR in a spatial or temporal sequence can provide another navigation strategy. In our body, leukocytes are rarely exposed to only one chemotactic factor. For efficient trafficking between organs and coordinated movement within tissues, most leukocytes express more than one GPCR on their surface and sense a wide range of chemokines and chemoattractants. Directional decision‐making in lymphoid and inflamed organs requires leukocytes to constantly sense, compare, and integrate numerous GPCR signals emanating from multiple sources. To efficiently navigate in a complex milieu of attractant gradients, leukocytes need to respond to combinational signals and prioritize between competing signals. While this aspect of GPCR signal integration has already been of interest decades ago, it has regained momentum with the recent developments in fabricating microfluidic‐based chemotaxis devices. We will now briefly summarize common mechanisms and concepts for leukocyte navigation, which become already relevant in the presence of a second GPCR signaling system (“Two‐GPCR systems”) (Figure 3). As leukocytes often use combinations of more than two‐GPCR systems, these principles could be further expanded. Manifold scenarios of attractant interpretation are conceivable, when leukocytes sense two chemoattractant signals through two different GPCR types (Figure 3). The outcome for leukocyte guidance depends on several determining parameters: (a) the spatial distribution of each signal (uniform, graded), (b) the spatial orientation of both signals toward each other (same or opposing directions), and (c) the ratio of signaling input that leukocytes receive from both signals (equal, unequal). The latter point includes both cell‐intrinsic features and cell‐extrinsic conditions. Unequal signaling input can result from different cell surface expression of the two‐GPCR types or different strength of the downstream signaling pathway. In both cases, one chemoattractant signal dominates over the other. This can also occur in leukocytes with equal signaling input from both GPCR systems under conditions of unequal concentrations of both signals in the extracellular space. Many of these scenarios have already been addressed with select leukocyte subsets and their cell‐specific GPCR combinations in controlled in vitro microenvironments, and examples will be discussed in the next paragraphs about specific leukocyte subsets (neutrophils, T cell, and DCs). As discussed before, homogenous chemoattractant fields can induce leukocyte polarity and chemokinesis. A second‐graded signal “on top” can then redirect prepolarized leukocytes and guide them toward other locations. However, if the uniform attractant field is very potent, it will retain leukocytes and attenuate chemotaxis toward the second‐graded signal (Figure 3A).45-48 Under conditions when both attractant signals are oriented in the same direction and overlap, the two‐GPCR signals have usually additive effects for leukocyte chemotaxis (Figure 3B). In the case of promigratory signals, chemotactic factors act often synergistically to improve chemotaxis along the same direction.49 Physiologically, this might be of particular relevance for tissue sites where overlapping attractant gradients originate from the same local area. A special variant of GPCR signal synergism has initially been observed in migrating human and mouse neutrophil populations in vitro. During their chemotaxis in response to shallow N‐formyl‐peptides (fMLF) gradients, neutrophils released the lipid leukotriene B4 (LTB4) that acted on top of the early chemotactic factor as a signal relay molecule to improve chemotaxis of the whole neutrophil population.50 Under conditions when leukocytes encounter two spatially opposing gradients, the two‐GPCR signals compete for guiding leukocytes into one or the other direction (Figures 3C and 4). Leukocytes exposed to two opposing attractants with equal signaling strength will likely accumulate and find migratory equilibrium in the middle of both gradients (Figure 3C).47, 48, 51, 52 However, an unequal signaling input from both signals favors the movement of leukocytes into one direction (Figure 4A).47 It is known for decades that specific GPCR signals can dominate over other GPCR signals, which enables leukocytes to prioritize some chemoattractant signals over others (Figure 4B).42, 45 This aspect of GPCR crosstalk provided a first conceptual framework for neutrophils traveling in a stepwise fashion from the blood into infected tissues.45 Initially, it was discussed that heterologous desensitization of GPCRs might be an underlying mechanism for prioritizing chemotactic cues. In contrast to homologous desensitization (see above), heterologous GPCR desensitization is agonist independent and results from the phosphorylation of one GPCR by a kinase activated through the signaling cascade of another activated GPCR (Figure 3D). Thus, one‐GPCR signal can act as desensitizing signal for multiple other GPCR types, causing leukocytes to become refractory to several chemotactic stimuli within minutes. This rapid cross‐phosphorylation has been linked to second‐messenger kinases, particularly protein kinase C, which phosphorylates the GPCR at sites different from GRKs.53 For neutrophil navigation in opposing gradients, it was later proposed that the dominance of certain GPCR signals is regulated by events downstream of the receptors. According to this concept, referred to as “signaling hierarchy”, special GPCRs induce intracellular signaling pathways that can override signaling pathways downstream of other GPCR signals42, 54 (see below for details). Leukocyte populations in tissues rarely consist of identical cell clones, but are rather characterized by many cells with slightly heterogeneous expression of GPCR combinations. Thus, single‐cell heterogeneity on the basis of combinatorial GPCR expression may influence the population response in two opposing attractants with equal signaling strength (Figure 4C). In many physiological settings, leukocytes are thought to navigate step‐by‐step through sequential chemotactic fields. Under conditions of two spatially segregated chemotactic signals in sequence (Figure 3E), leukocytes may first move along a first attractant gradient (Figure 1A), and then subsequently sense the gradient of a second, more distant attractant. During the transition from first to second attractant gradient leukocytes then encounter a competitive situation between two gradients (Figure 3C). When the second signal is dominant over the first one, it is plausible that leukocytes are recruited to and then retained at the distant site. Importantly, even without any dominance between the signals, leukocytes can migrate away from a first attractant and travel toward a second, more distant attractant.45 In principle, this also allows leukocytes to leave chemotactic fields of high, saturating concentrations. Moreover, leukocytes remain responsive for additional attractant changes, while moving back and forth in the equilibrium between both gradients. Thus, interpreting two distinct signals in sequence allows efficient navigation patterns that are not possible with only one attractant presented in the same spatial array. We want to point out that the sequential sensing of attractant gradients can also be temporally controlled (Figure 3E). Leukocytes may undergo sensing of a first attractant gradient, while a second attractant arises newly in close vicinity. In the course of an immune response in lymphoid or inflamed tissues, this may result from chemoattractant secretion of nearby activated tissue‐resident cells or recently recruited, blood‐derived leukocytes. As most immune responses are choreographed by a sequence of timely separated cellular events, it is likely that this aspect of sequential sensing has strong influence on leukocyte navigation in situ. In our contemplation on leukocyte navigation by “Two‐GPCR systems”, we have focused until now on GPCRs with signaling function. However, many leukocyte subsets additionally express members of the atypical chemokine receptor (ACKR) family on their surface. Also referred to as scavenging receptors, ACKRs are GPCRs with promiscuous binding affinity for multiple chemokines. As they do not couple to G‐proteins, they are unable to induce intracellular signaling pathways. Instead, ACKRs can actively internalize and target chemokines for lysosomal degradation.55 Functional co‐expression of conventional chemokine receptors and ACKRs in leukocytes has been implicated in the fine‐tuning of leukocyte chemotaxis, with ACKRs acting as rheostats in restricting excessive migration in response to moderate chemokine levels (Figure 3F).56-58 As we have just outlined, leukocytes have evolved several molecular mechanisms that allow an enormous spectrum of GPCR‐controlled navigation strategies. Many of the emerging concepts for leukocyte navigation in complex chemoattractant fields have been developed in controlled in vitro systems, some of which by studying only one select leukocyte subset. Rigorous testing and proving the relevance of these concepts for leukocyte migration in the complexity of mammalian organs and tissues is technically very challenging. However, systematic studies in mice have started to delineate GPCR‐controlled navigation strategies and their influence on physiological trafficking patterns through lymphoid organs and inflamed peripheral tissues. In the following, we will discuss our current knowledge on the role of GPCR signals for the migration patterns of three leukocyte subsets (neutrophils, T cells, and DCs). Each of these immune cell types has evolved their own trafficking paths to fit their effector functions. We will highlight recent studies that conceptually advanced our understanding of GPCR‐controlled leukocyte navigation in mammalian tissues. 2 NEUTROPHILS Neutrophils are pivotal effector cells of our innate immune response and the first line of defense for eliminating bacteria and fungi in tissues.59 Recent studies also implicate regulatory functions of neutrophils in tissue homeostasis and cancer.60 In mammals, they are produced in the bone marrow and are continuously released into the bloodstream to patrol the intravascular compartment under homeostasis. Neutrophils circulate in the blood for ca. 12 hours under the influence of circadian rhythms, before they are mostly cleared into the bone marrow, but also other extramedullary tissues.61 In contrast to the general believe that naive peripheral tissues are free of neutrophils, recent work suggests that low numbers of neutrophils are present in many tissues under steady state.62 Upon inflammation and infection, the release of neutrophils from bone marrow increases their concentration in the blood by several folds.63 Moreover, large numbers of circulating neutrophils then infiltrate inflamed tissues where they sense activated vascular endothelium. In agreement with their critical role as first responders, neutrophils have evolved robust strategies to infiltrate inflamed tissues extremely fast and orient themselves to detect potential sites of microbial invasion with great precision. In many organs, the exit from the vascular compartment involves a sequence of exquisitely coordinated processes: (a) intravascular arrest, migration, and crawling along the activated endothelial cells, and (b) breaching multiple layers of the vascular wall (venular endothelium, venular basement membrane, and pericytes).64 Once neutrophils have traversed this barrier, they are primed for the rapid detection of damaged tissue or microbes in the extravascular space. At sites of cell death in the interstitial space, neutrophils undergo sequential phases of highly coordinated chemotaxis and cell cluster formation, now commonly referred to as neutrophil swarming.65, 66 As true sentinel cells, they sense almost any form of tissue insult, rapidly localize it in any inflamed organ and efficiently move toward the core of the inflammation or infection site. Neutrophils have been shown to express on their cell surface more than 30 GPCR types, which can all bind chemotactic molecules. Accordingly, neutrophils are responsive to a broad array of chemically diverse chemoattractants, including chemokines, lipids, complement factors, formylated peptides, nucleotides, alarmins, and proteolytic (ECM) fragments.67-69 Many of these chemotactic factors are usually present at sites of in vivo inflammation. How the crosstalk between these GPCR signals guides neutrophil recruitment in inflamed tissues has been of huge interest since decades. Early in vitro work with human polymorphonuclear neutrophils (PMN) characterized the strength of chemically distinct chemoattractant classes to desensitize calcium‐ion mobilization, as a read‐out of GPCR downstream signaling, to one another (Figures 2D and 3D).53, 70 Peptide chemoattractants, including N‐formylmethionyl‐leucyl‐phenylalanine (fMLF), the complement factor C5a, and the chemokine CXCL8 (IL‐8), cross‐desensitized one another to different extents (fMLF > C5a > IL‐8). Moreover, peptide chemoattractants, in particular fMLF, cross‐desensitized lipid chemoattractants, including platelet‐activating factor (PAF) and LTB4. In contrast, lipid and purinergic agonists failed to desensitize to the peptide chemoattractants.70 These results were extended to neutrophil chemotaxis in the presence of multiple chemoattractants, showing a hierarchy of resistance to competing signals, with fMLF and C5a inhibiting migration to other chemoattractants (Figure 3D).71, 72 Elegant under‐agarose chemotaxis experiments proposed the concept that neutrophils can navigate through complex chemoattractant fields by migrating in a step‐by‐step manner in response to one agonist source after another. Thus, their ultimate positions are determined combinatorially by the array of GPCRs they express and by the sequence of chemoattractant gradients they encounter (Figure 3E).45 In this concept, pathogen‐derived “end‐target” chemoattractants (N‐formyl peptides, C5a) will dominate over host tissue‐derived “intermediate‐target” chemotactic factors (CXCL8, LTB4) in their ability to guide neutrophil migration (Figure 3A, C, E).45 This dominance of end‐target attractants has been suggested to depend on the activation of the p38 MAPK pathway, which can override the phosphoinositide 3‐kinase (PI3K) pathway activated by intermediate‐target attractants.42, 54 Importantly, neutrophil navigation is also possible in a spatially sequential array of intermediate‐target chemoattractants (Figure 3E).45, 47, 51, 73 Another aspect of crosstalk between end‐target and intermediate‐target chemoattractants has not been considered in these early studies, but is also functionally relevant for neutrophil navigation. Already small amounts of fMLF or C5a can stimulate neutrophils to secrete intermediate‐target attractants, in particular LTB4.50, 74 This paracrine signaling mechanism becomes very relevant in chemotaxing neutrophil populations. Neutrophil‐released LTB4 functions as a signal relay molecule and amplifies in a feed‐forward manner the chemotactic response to subsaturating concentrations of end‐target attractants (Figure 3B).50, 75 Recent developments in microfabricated devices and microfluidic‐based chemotaxis systems now allow a more detailed analysis of neutrophil migration responses in complex chemoattractant milieus, providing deeper insight into neutrophil navigation in opposing gradients (Figure 3C),47, 73, 76-78 synergistic gradients (Figure 3B),49 and geometrically complex environments.49, 77 Moreover, even the analysis of GPCR signaling underlying neutrophil swarming dynamics is now possible due to novel micropatterning techniques.75 As human PMNs, mouse neutrophils express a range of functional GPCRs to sense N‐formyl peptides (through FPR1 and FPR2), C5a (mainly through C5ar1), LTB4 (mainly through LTB4R1/BLT1), and several chemokines including CXCL1, CXCL2, CXCL5 (through CXCR2), CXCL12 (through CXCR4), and several CC‐chemokines (through CCR1).67 Some of these GPCRs are present in secretory vesicles that can rapidly fuse with the plasma membrane upon neutrophil activation, allowing the cells to increase their responsiveness to chemotactic cues (Figure 2A).38 Other reported mechanisms to control GPCR functionality in neutrophils include competitive antagonism or partial agonism (Figure 2C),79 receptor multimerization (Figure 2B), differential agonist responses (Figure 2E),80 and agonist scavenging through simultaneous expression of ACKRs (Figure 3F).56 Homologous desensitization is a well‐known phenomenon for almost all neutrophil‐expressed GPCRs (Figure 2D). While GPCR transducers (G‐protein subunit types, GRKs, and arrestins) and RGS proteins have been linked to the function of some GPCRs in primary mouse neutrophils,32, 81-88 we are still missing a detailed understanding of their exact contributions to neutrophil navigation in complex chemoattractant fields. Clearly, neutrophils are equipped with many GPCR types and mechanisms to dynamically control GPCR activity, allowing them to use various navigation strategies in the presence of multiple chemotactic cues. Moreover, neutrophils have been shown to perform fugetaxis against the direction of a gradient of high concentration chemokine, a process that is mechanistically not well understood (Figure 1C).89 However, there are still many open questions of how these mechanisms contribute to the neutrophil migration patterns observed in situ. Intravital microscopy studies of GPCR‐deficient neutrophils provided novel insight into how GPCR signals choreograph the physiological trafficking paths of neutrophils. In the following, we will point out that GPCR signals are important throughout many steps in the lifecycle of a neutrophil. In particular, we will highlight studies that advanced our concepts of GPCR‐controlled neutrophil navigation in mouse tissues. 2.1 Bone marrow release: inhibiting chemokine‐mediated retention The majority of mature neutrophils in the body reside in the bone marrow, with an estimated ratio of 300:1 between neutrophils in the bone marrow and blood. Neutrophil release into the blood is controlled by the interplay of retention and mobilization signals, provided by two antagonistically operating chemokine systems (Figure 3A,C). During homeostasis, the chemokine CXCL12 is produced in the bone marrow and essential for the retention of mature neutrophils that express the corresponding chemokine receptor CXCR4.90 CXCL12 is expressed at highest levels by “CXCL12‐abundant reticular cells” (CAR cells), but also by other stromal cells of the bone marrow, including osteoblasts.63, 91 The expression levels of CXCL12 in stromal cells follow a circadian rhythm, controlled by the sympathetic nervous system.92 When neutrophils were freshly isolated from the bone marrow, they expressed only low levels of cell surface CXCR4, but contained high intracellular levels of CXCR4.90, 93 After placing these cells in medium lacking CXCL12, they upregulated cell surface CXCR4 within hours.90, 93 These findings suggested receptor desensitization and internalization in response to constitutively high local concentrations of CXCL12 in the bone marrow microenvironment (Figure 2D). Moreover, these data underlined that neutrophils receive constant CXCL12 signals, which position them in the bone marrow, probably by activating integrin‐mediated adhesion to stromal cells.94 While high concentrations of CXCL12 retain neutrophils in the bone marrow via CXCR4 signaling, low concentrations of CXCR2 ligands expressed on endothelial cells and produced by megakaryocytes facilitate neutrophil egress via CXCR2.63 Thus, CXCR2 signals recruit neutrophils out of the retentive CXCL12 field and direct them to the endothelial sinus walls for entry into the blood circulation (Figure 3A). This intricately balanced interplay between retention and mobilization signals can be shifted in both directions. G‐CSF treatment or acute inflammation, which increases blood serum levels of G‐CSF and CXCR2 ligands, shifts the balance toward neutrophil mobilization from the bone marrow.95-98 Several modes of action have been proposed for G‐CSF, either leading to the lowering of CXCL12 levels in the bone marrow or triggering the release of CXCR2 ligands from endothelial cells or megakaryocytes (summarized in63). The same shift toward neutrophil egress can be achieved by conditional gene depletions of CXCR4 or CXCL12 in mice.99, 100 The administration of the CXCR4 antagonist AMD3100 also results in a threefold increase in the number of circulating neutrophils and a corresponding decrease in bone marrow numbers.93, 101 Originally, this drug effect was interpreted as direct antagonism of CXCR4 expressed by neutrophils, thereby disrupting the CXCL12‐CXCR4 axis in the bone marrow (Figure 2C).90 Surprisingly, subsequent studies found that AMD3100 also acted on CXCR4 expressed by bone marrow endothelial cells, and inhibited the translocation of CXCL12 across bone marrow endothelial cells, suggesting a rapid rise of CXCL12 in the blood and reversal of the CXCL12 gradient (Figure 3B).102, 103 These data were questioned by intravital imaging studies that could not observe clear neutrophil egress from the bone marrow upon AMD3100 treatment. Instead, the increase in circulating neutrophils through AMD3100 was explained by the release of marginated neutrophils from the lung microvasculature.104 Moreover, AMD3100 had a blocking effect on the trafficking of aged blood neutrophils, which upregulate CXCR4 expression over time, enabling them to home back to the bone marrow for clearance.104 The exact mode of action of AMD3100 is still controversially discussed.90 Insight into the physiological consequences of shifting the chemokine balance in the opposite direction, toward retention in the bone marrow, comes from an extremely rare congenital human immunodeficiency disease. Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an autosomal dominant human disorder caused by gain‐of‐function mutations in the C‐tail of CXCR4.105, 106 WHIM syndrome involves myelokathexis, a condition characterized by the retention of mature neutrophils in the bone marrow and peripheral neutropenia. WHIM‐associated truncations or mutations in the carboxy‐terminal tail of CXCR4 result in the loss of multiple serine residues, which are target sites for GRK‐mediated receptor phosphorylation. Truncated forms of CXCR4 are impaired in agonist‐induced homologous desensitization and internalization, and cause enhanced signaling upon CXCL12 stimulation.106 This leads to stronger anchorage of mature neutrophils in the bone marrow, counteracting CXCR2‐mediated chemotaxis toward vessels and neutrophil egress. However, administration of G‐CSF or ADM3100 to WHIM patients can overcome this imbalance and cause an increase in circulating neutrophil numbers.106 2.2 Getting to the site of inflammation: navigation along multiple chemokine fields Blood‐circulating neutrophils detect sites of tissue injury and infection by interacting with the luminal surface of activated venular endothelium. In response to tissue‐released inflammatory cytokines, endothelial cells upregulate P‐selectin, E‐selectin, and integrin ligands, such as ICAM‐1 and VCAM‐1, to promote interactions between neutrophils and the vessel wall. This induces neutrophils to undergo a multistep adhesion cascade along the endothelial lining before emigrating from the vessel into the inflamed tissue interstitium.107 Chemokines are present on the luminal surface of endothelial cells and cause arrest of rolling neutrophils by triggering the activation of integrins. Many studies showed an involvement of CXCR2 ligands, in particular CXCL1, but also CXCL2 and CXCL5, in this process.108, 109 These chemokines are primarily expressed and released by endothelial cells,110, 111 but also tissue‐resident cells, including pericytes, perivascular macrophages, and mast cells, can act as important chemokine sources.112-115 Due to their high‐affinity binding sites for heparin sulfate (HS) GAG most chemokines become immobilized by HS GAG on the luminal side of the endothelium,116 and defects in HS GAG synthesis have been shown to decrease immobilization of chemokines and neutrophil transmigration into inflamed tissues.117, 118 While the involvement of chemokines for neutrophil transmigration is widely established, there has been ongoing debate whether and how leukocytes sense stable chemokine gradients across the vessel wall on their way into the tissue (Figure 1B). There are ample references to support the common believe that chemotactic gradients between the apical and basolateral part of postcapillary venules promote diapedesis, ie, the outward passage of neutrophils through the vessel wall. Immunofluorescence stainings of HS GAG moieties revealed steep gradient of HS GAG scaffolds between the luminal and abluminal aspect of skin postcapillary venules.119 Under inflammatory conditions, HS GAG distribution became even more polarized by massive depositions of HS and laminin in the basolateral basement membrane of skin vessels.119 Given the GAG‐binding properties of many chemokines, it was proposed that these sharp HS GAG scaffold gradients might favor chemokine gradients across the venular wall. In agreement with this idea, excessive deposition of the GAG‐binding chemokine CXCL1 on the apical part of inflamed blood vessels resulted in decreased neutrophil diapedesis.120 Other supporting data come from experiments in which an extravascular source of CXCR2 ligands was placed in form of a gel on one side of a blood vessel.121 Under these conditions, neutrophil crawling was directed to emigrate preferentially from that side of the vessel. HS GAG on the surface of the endothelium was required to present CXCL2, forming a chemotactic gradient that was followed by neutrophils. This sequestration of endothelial chemokine occurred exclusively in venules, but not other blood vessel types.121 HS GAG, together with the atypical receptor ACKR1, was also discussed to establish gradients by transcytosis of chemokines.118, 122, 123 Transcytosis involves the internalization and transport of tissue‐derived inflammatory chemokines from the basal surface onto the luminal surface of endothelial cells. ACKR1 is specifically expressed by postcapillary venules and was shown to support promigratory chemokine patterns across the vessel wall for optimal leukocyte migration across the vessel wall.123, 124 Unlike other ACKR, ACKR1 does not act as classical scavenger receptor, and was initially discussed to promote the transcytosis of tissue‐derived inflammatory chemokines from perivascular pools to the apical surface of venules.122, 123 Novel insight into the functional role of ACKR1 and the directional cues that guide neutrophils during diapedesis come from recent elegant experiments of Nourshargh and colleagues.125 By studying TNF‐induced neutrophil emigration in the inflamed mouse cremaster muscle, it was shown that neutrophils encounter the two CXCR2 ligands CXCL1 and CXCL2 in a sequential manner during their passage through the vessel wall. In a three‐step process, neutrophils first sense CXCL1 produced by TNF‐stimulated endothelial cells on the luminal surface of venules, promoting crawling along the endothelium. Next, neutrophils encounter CXCL2 that is retained at endothelial cell junctions by ACKR1. Notably, CXCL2 is secreted by neutrophils themselves and then presented by ACKR1, which is enriched in endothelial junctions. This self‐guided migration response of neutrophils was critical for breaching of endothelial junctions and unidirectional transendothelial migration. Lastly, CXCL1 expressed by pericytes governed neutrophil crawling in the subendothelial space.125 While earlier work indicated relatively restricted expression of CXCL1 to vascular and tissue‐resident cells, and CXCL2 with activated neutrophils,126 these novel findings highlight unexpected non‐redundant roles for CXCL1 and CXCL2 to guide neutrophils through venular walls as governed by their distinct cellular sources.125 Having identified this critical spatiotemporal sequence of chemokine‐guided steps across the vessel wall (Figure 3E), it will be of future interest to understand how other tissue‐derived attractant signals may act on top of this and support further guidance into the underlying inflamed interstitium. As an example, neutrophils entering an inflamed peritoneum were suggested to encounter two sequential gradients in a step‐by‐step manner (Figure 3E).114 In a lipopolysaccharide‐induced peritonitis model, initial recruitment of neutrophils out of the blood vessel into the peritoneal tissue was regulated by rapid secretion of CXCL1 and CXCL2 from perivascular mast cells. Further guidance of extravasated neutrophils through the interstitium and into the peritoneal cavity depended on CXCL1 and CXCL2 produced by tissue‐resident interstitial macrophages.114 2.3 Getting to the site of inflammation: interpreting temporal cascades of chemically distinct chemoattractants Most of our discussions until now focused on the role of chemokines for neutrophil guidance in the tissue, but also putative non‐GAG‐binding, soluble chemoattractants including C5a, LTB4, and formylated peptides are sensed by neutrophils and relevant for special types of inflammatory reactions. A mouse model of autoimmune rheumatoid arthritis, the K/BxN serum transfer model, has provided unprecedented insight into how four different chemoattractant receptors, C5aR1, LTB4R1, CCR1, and CXCR2, collaborate to recruit neutrophils into the inflamed tissue.127 In this model, the inflammatory response is driven by immunoglobulin G autoantibodies to the ubiquitous glycolytic enzyme glucose‐6‐phosphate isomerase (GPI) leading to the formation of immune complexes that drive immune cell activation. Neutrophil infiltration into the joint is critical for the clinical signs of arthritis in this model. Several consecutive studies have elucidated a temporal cascade in which neutrophils encounter complement, lipid, and chemokine signals to enter the joint and contribute to disease progression (Figure 3E).74, 128-131 While it has long been known that immune complexes lead to the activation of the alternative complement system in arthritis,132, 133 it has only recently been found that C5aR signaling in circulating neutrophils is the key initiator for a cascade of events that ignite inflammation.131 The deposition of immune complexes in the joint space induces the generation of C5a, which unexpectedly binds to HS GAG on the synovial endothelium. Neutrophils sense C5a on the joint endothelium, induce integrin‐dependent arrest, and facilitate crawling along the vasculature. Subsequently, C5a signals stimulate neutrophils to secrete LTB4, which induces the entry of the first neutrophils into the joint and initiates the onset of disease.74, 128, 129, 131 After disease onset, recruited neutrophils in the joint secrete IL‐1β, which in turn stimulates fibroblast‐like synoviocytes to produce CCR1 ligands at first and later CXCR2 ligands.130 CCR1 ligands are required to recruit the next wave of neutrophils into the joint, and this recruitment of neutrophils is broadly amplified in the last step of the cascade when neutrophils themselves release CXCL2 to potently attract large numbers of neutrophils into the joint.67, 130 Thus, neutrophil entry into the inflamed joint is a prime example where neutrophil recruitment is choreographed by non‐redundant roles of distinct chemoattractants that collaborate sequentially in a temporal cascade (Figure 3E). Moreover, this process highlights the rapid production of complement and LTB4 to initiate a recruitment cascade at the local site of inflammation. By contrast, chemokines act later in this cascade to amplify and prolong the inflammatory response.67 2.4 At the site of inflammation: synergism through lipid‐mediated amplification Another example where complement‐induced LTB4 secretion rapidly shapes neutrophil migration patterns has recently been described.134 In a simplified fungemia mouse model, the fungi Candida albicans sequestered in the pulmonary vaculature and actively recruited neutrophils. Neutrophils showed swarm‐like migration patterns in response to sequestered yeast, similar to previously described swarming responses in other tissues.66 A small number of lung neutrophils responded within minutes and performed intravascular chemotaxis, before larger numbers of neutrophils were recruited and formed cell clusters. The immediate capture of live C. albicans by neutrophils depended on complement‐mediated chemotaxis.134 In addition, complement activation stimulated neutrophils to secrete LTB4, which amplified the directional recruitment and clustering of neutrophils in a synergistic manner (Figure 3B). This was in agreement with a previous study highlighting the importance of LTB4‐mediated signal amplification during neutrophil swarming in interstitial tissue spaces at sites of sterile skin injury and in bacteria‐infected lymph nodes.135 Neutrophil swarming is an essential process of the neutrophil tissue response and has been observed in diverse tissues under conditions of sterile inflammation and infection with various pathogens, including bacteria, fungi, and parasites.65, 66 The swarming response comprises sequential phases of highly coordinated chemotaxis followed by neutrophil accumulation and the formation of substantial neutrophil clusters, which has led to the multistep attraction model of neutrophil swarming.66 While the underlying signals and molecular players for some of these steps are still unclear, we know that neutrophil‐derived LTB4 acted on top of early released, yet unknown, chemoattractants and mediates intercellular signal relay among neutrophils to amplify interstitial recruitment in a feed‐forward manner (Figure 3B). This model was in accordance with earlier in vitro studies identifying LTB4 acted as a signal relay molecule (see above).50 As a consequence, LTB4‐LTB4R1 signaling enhances the radius of neutrophil recruitment from distant tissue sites and maintains the amplified chemotactic response of the neutrophil population. Together with signaling through other GPCRs, such as CXCR2 and FPR2, LTB4R1 signaling also optimizes neutrophil clustering.135 Importantly, LTB4 and CXCR2 signals are also relevant for the swarming response of human neutrophils.75 The formation of focalized dense neutrophil aggregates is considered beneficial for isolating sites of tissue wounding from viable surrounding tissue, containing microbial invaders and concentrating the microbicidal activity of a whole neutrophil population in one swarm center.66 However, neutrophil aggregation can also lead to tissue damage and removal of other cells.135, 136 In the context of the lung microvasculature, intravascular neutrophil swarms that formed in response to C. albicans infection lead to vessel occlusion and pulmonary hemorrhages, which were reverted in the absence of LTB4R1.134 How neutrophil‐derived LTB4 shapes and maintains a promigratory chemotactic field in vivo remains unclear. LTB4 is considered to have a short half‐life and can be rapidly turned into LTB4 metabolites, which act as natural inhibitors of LTB4‐mediated responses. However, it has also been demonstrated that neutrophils release LTB4 in form of exosomes, which may support more prolonged effects of LTB4 gradients.137 Interesting insights into this issue come from studies with diabetic mice receiving skin infections with methicillin‐resistant Staphylococcus aureus (MRSA), which leads to large neutrophil clusters in form of abscesses.138 Diabetic mice produce higher levels of LTB4 in the infected skin compared to non‐diabetic mice. Surprisingly, neutrophils in the skin of these mice failed to assemble into an organized abscess, as excessive LTB4 levels likely disrupted chemoattractant gradients in the skin and impaired directed neutrophil chemotaxis.138 These findings exemplify that the appearance or absence of neutrophil swarms depends on the specific mixture of chemoattractants in a specific inflammatory environment. Although not yet studied in detail, we know that several factors, including tissue damage size, presence of pathogens, induction of cell death, and neutrophil numbers, are important for shaping the exact neutrophil‐swarm phenotype in an inflamed tissue. All these factors can potentially initiate the release of chemotactic fields that may strengthen or attenuate LTB4‐mediated signal amplification in situ.66 2.5 Getting to the site of inflammation: following hierarchies True prioritization of chemoattractant fields has been observed for neutrophils in a model of thermal‐induced liver injury.139 In this model, large numbers of neutrophils are recruited over hours from the liver sinusoids to a focal site of sterile hepatic necrosis. The necrotic area releases several molecules that shape the adjacent hepatic environment and ultimately form two distinct chemotactic fields at different distances from the necrotic focus. During their search for damaged tissue neutrophils sequentially navigate through these two chemotactic fields. First, neutrophils follow an intravascular gradient of GAG‐bound immobilized CXCL2 that supports integrin‐dependent crawling toward the necrotic focus. This CXCL2 haptotactic gradient abruptly halts, approximately 150 μm proximal to the border of the necrotic tissue. Second, neutrophils at this border continue to migrate on top of platelets along non‐perfused vasculature, before entering the necrotic area.139, 140 When neutrophils were lacking the formyl‐peptide receptor FPR1, they migrated along the healthy sinusoids to the highest concentration at the end of the haptotactic CXCL12 gradient and never entered into the platelet‐rich and following necrotic zone. Given the important role of FPR1 signaling during this process, mitochondrial‐derived formyl peptides released from dying hepatocytes were suggested to attract neutrophils into the necrotic area. Of interest, FPR1 signaling has also been shown to regulate neutrophil recruitment into necrotic areas of hepatic ischemia‐reperfusion injury.141 In contrast to formyl peptides released from bacteria, endogenous formyl peptides are produced by mitochondria and become released upon cellular damage.142 Similar to bacterial formyl peptides, also mitochondrial formyl peptides can promote neutrophil chemotaxis in an FPR‐dependent manner.139, 142 Thus, neutrophil navigation in the liver injury model follows a spatial cascade of chemoattractants (Figure 3E) and appears to depend on the prioritization of an end‐target attractant (eg, fMLF) over an intermediate‐target attractant (eg, CXCL2) (Figure 3A). Recent elegant work by Kubes and colleagues revealed that neutrophils play a critical role in fully repairing the hepatic injury. In the course of this process, neutrophils at the border of the injury left the injury site and reentered healthy liver sinusoids.143 However, it remains to be shown if GPCR‐controlled mechanisms are involved in this form of reverse migration into the vasculature. 3 T CELLS T cells and B cells represent the cellular hallmark of the adaptive immune system. These lymphocytes are generally long‐lived cells and are driven by GPCR‐mediated guidance throughout their life cycle. Being released from the bone marrow as a common lymphoid progenitor, T‐cell precursor settle in the thymus where they tread a highly regulated developmental path, guided by GPCRs. Once T cells have been fully matured and selected, they exit the thymus and commence their recirculation pattern between secondary lymphoid organs (SLO). As the T‐cell receptor (TCR) repertoire needs to be highly diverse in order to raise the chance to efficiently detect foreign antigens against all kind of pathogens, the naive T‐cell precursor frequency for a specific antigenic determinant is very low (≈1/105‐1/106). Consequently, during a newly arising infection, antigen‐presenting DC need to interact with millions of T cells in order to activate the few rare naive T cells that optimally react to the foreign antigenic determinant. To facilitate this process T cells need to efficiently migrate between and navigate within SLOs to optimize such DC encounters. Once T cells are activated and primed by a DC carrying and displaying the proper antigen, they differentiate and change their GPCR expression pattern in order to leave the SLO and enter infected tissues via the blood stream. Here, GPCRs pilot them toward infected cells where they execute their specific effector function in order to eliminate or at least control the microbial invasion. In vitro experiments to address how lymphocytes respond to chemokines were primarily based on transwell, under agarose assays or 3D ECM‐like gels, showing that chemokinesis, chemotaxis, and homologous and heterologous desensitization apply to GPCR‐mediated navigation of T cells.144-149 However, these approaches have limitations when trying to address how T cells respond to more complex chemokine fields involving multiple GPCRs. With the development of microfluidic devices150 and more recently, microfluidic mazes, T‐cell exploratory patterns and chemotaxis in response to chemokines could be analyzed in more detail, while providing the investigator with a higher level of control and precision.151 Overall, however, there are only a few studies that have addressed complex GPCR interactions in T cells in vitro. In the following sections we will focus on what we have learned from in vivo experiments regarding the trafficking of T cells. 3.1 Searching for the antigen: exploration and compartmentalization In vivo, chemo‐ and haptokinesis are particularly prevalent in lymphoid organs and underlie the continuous undirected or random‐walk migratory activity of lymphocytes (Figure 1A).9 Functionally, this migratory behavior is required to enable encounters and consecutive interactions between leukocytes. In other words it reflects the search strategy of lymphocytes to detect rare interaction partners in a large and densely populated three‐dimensional space. Given the low precursor frequency of ly