1 INTRODUCTION

To fulfill their function of immune defense and surveillance, most leukocytes are not stationary positioned in the body but constantly migrate within tissues or circulate between tissues and organs.1, 2 Considering that active interstital migration is very slow, leukocytes use blood and lymphatic vessels to rapidly move between different sites of the body. Over the past decades, the process of immune cell migration out of blood vessels has been studied in great detail, culminating in the identification of the intravascular adhesion cascade and a plethora of molecules involved in leukocyte extravasation from different blood vascular beds in steady-state and in inflammation.3-5 More recent technologic advances in intravital imaging, particularly the use of confocal and two-photon microscopy, have opened up new ways of studying leukocyte interstitial migration and immune function in tissues like the skin, lymph nodes (LNs) or lung.2, 6, 7 Compared to leukocyte extravasation from blood vessels, migration through lymphatic vessels is much less well explored. This is likely due to the fact that the lymphatic vasculature as a whole has been far less well studied in comparison to the blood vasculature. In fact, the first markers allowing to faithfully distinguish blood vessels from lymphatic vessels only emerged a bit more than 20 years ago. Ever since research on lymphatic biology has exploded and there is a growing interest in this vessel type and its involvement in health and disease.8-10 Considering that one of the main functions of lymphatic vessels consists in mediating the migration and transport of leukocytes, it is not surprising that recent years have witnessed great advances in our knowledge of lymphatic trafficking. Besides deciphering the mechanisms of lymphocyte egress from LNs into efferent lymphatics, we have also gained a good mechanistic understanding of how dendritic cells (DCs) and T cells – that is, the main cell types found in afferent lymph - migrate into and within afferent lymphatic vessels. In this review, we will report on how imaging has contributed to deciphering the cellular and molecular determinants of leukocyte trafficking through afferent lymphatics. We will first introduce the biology of afferent lymphatics and report on the importance of DC and T cell migration for immune function. Next, we will focus on selected recent discoveries in the field of DC and T cell trafficking via afferent lymphatics that have been achieved by time-lapse imaging and discuss their presumed relevance for the induction and regulation of the immune response.

2 STRUCTURE OF THE AFFERENT LYMPHATIC VASCULATURE IN THE SKIN

The lymphatic system is composed of primary and secondary lymphoid organs (SLOs) and lymphatic vessels. The lymphatic vasculature comprises a hierarchically organized network of vessels that can be subdivided into three subgroups, based on their localization in tissues; afferent lymphatics, which connect peripheral tissues with draining LNs (dLNs), the lymphatic vasculature present within LNs and the efferent lymphatics, which exit from LNs.8-10 Similarly to blood vessels, afferent lymphatic vessels are present in virtually all tissues of the body. They originate as blind-ended structures, so called capillaries, which merge into a second type of vascular bed, that is, the collecting vessels. The latter subsequently fuse into even larger collectors which leave the tissue and connect with the subcapsular sinus of dLNs. Within the LN, the various LN sinuses are lined by different types of lymphatic endothelial cells (LECs) with distinct gene expression patterns and function.11 Typically, one efferent lymphatic vessel leaves the LN and connects it to a further downstream-located LN. As LNs are frequently arranged in chains, the efferent lymphatic exiting from one LN can at the same time be the afferent collecting vessel of another LN located further downstream in the chain. Notably, in this review we will exclusively mean lymphatic vessels originating in peripheral tissues when talking about “afferent lymphatics”. Ultimately, larger efferent collectors converge in the central region of the body to form the thoracic and lymphatic ducts. The latter fuse with the subclavian veins and release the lymphatic content – that is, a cell-, protein- and lipid-rich fluid commonly referred to as lymph - into the blood circulation.

Afferent lymphatic vessels play a crucial role in the regulation of interstitial fluid homeostasis, as they return excess tissue fluid that has leaked out of blood vessels back to the circulation. Moreover, they are important for the uptake of dietary fats and vitamins from the intestine.8, 9 Afferent lymphatics further transport soluble antigen, inflammatory mediators as well as leukocytes from peripheral tissues to dLNs, establishing their crucial role in adaptive immunity. Much of our current knowledge of lymphatic morphology has been gained from old electron microscopy studies12, 13 and more recently from the analysis of tissue whole-mounts prepared from murine skin or trachea by confocal microscopy.14, 15 The latter studies have revealed that LECs present in lymphatic capillaries have a characteristic oakleaf shape14 (Figure 1). Peculiarly, capillary LECs partially overlap and are only loosely connected by button-like cell-cell junctions at the sites of such overlaps, thereby generating characteristic overhanging flaps. These flaps, also called primary valves, were first identified as the sites of leukocyte entry into lymphatic capillaries by electron microscopy.14 Originally described in the murine skin and trachea,14 the oakleaf shape of capillary LECs and button-like cell-cell junctions of afferent lymphatic capillaries have meanwhile been confirmed in many other murine tissues and also in human tissues like the skin.16, 17 Capillary LECs are further connected to the extracellular matrix (ECM) by anchoring filaments that force the vessel to expand and the flaps to open and fluid to enter when pressure increases, for example, during tissue inflammation18, 19 (Figure 1). Lymphatic capillaries are also surrounded by a discontinuous thin basement membrane (BM), which is composed of layers of a specialized sheet-like ECM, that forms the supporting structure of the endothelial cells. The lymphatic BM of both capillaries and collectors is primarily composed of laminin, type IV collagen, nidogens, and perlecan.15

Morphology and structure of afferent lymphatics. Afferent lymphatics begin as blind-ended capillaries present in peripheral tissues. Capillaries are composed of oakleaf-shaped LECs that are connected by discontinuous button-like cell-cell junctions. These junctions create open flaps between neighboring LECs that facilitate leukocyte entry and uptake of fluids and macromolecules. LECs also possess anchoring filaments that connect to the ECM and regulate the opening of the flaps. Capillaries are further surrounded by a thin and fenestrated BM. Lymphatic capillaries subsequently merge into collecting vessels. In contrast to the capillaries, LECs in collecting vessels are elongated and tightly connected to each other by zipper-like cell-cell junctions. Collectors are surrounded by a thick BM as well as LMCs. Collectors further have valves, which upon vessel contraction support unidirectional fluid flow toward the dLN

Conversely, LECs in collecting vessels, that is, the more downstream-located vessel segments, display an elongated shape and are surrounded by continuous, zipper-like cell-cell junctions, resembling the junctional setup of blood vessels (Figure 1). Due to the LEC-surrounding tight junctions, collecting vessels are much less permeable compared to capillaries and hence more suited for fluid containment and transport. Collectors are also surrounded by a similar yet in comparison to capillaries thicker and less fenestrated BM and by lymphatic muscle cells (LMCs), which confer contractility.20 Similar to veins, lymphatic collectors also contain valves that prevent fluid backflow upon vessel contraction and support lymph transport14, 21 (Figure 1). Because of the tree-like, hierarchical organization of the lymphatic network, with many capillaries merging into few collecting vessels and vessel contractions occurring in lymphatic collectors, also the lymph flow is markedly increased in this segment of the lymphatic vascular bed: While lymph flow in capillaries ranges from 1 to 30 μm/s,22, 23 velocities of up to several mm/s can be reached in larger collectors as, for example, in the mesentery.24, 25

3 LYMPHATIC MARKERS

The discovery of different lymphatic markers approximately 20 years ago has propelled lymphatic research and also facilitated the development of different molecular imaging approaches. These markers nowadays allow to unambiguously distinguish lymphatics from blood vessels or to distinguish between lymphatic capillaries and collectors. All LECs express the prospero-related homeobox 1 (Prox1), a master transcriptional regulator of lymphatic differentiation and identity and the receptor tyrosine kinase vascular endothelial growth factor (VEGF) receptor–3 (VEGFR-3) the receptor of the lymphatic growth factor VEGF-C. While podoplanin (also known as gp38), a mucin-type transmembrane protein, is also expressed by all LECs, the hyaluronan receptor LYVE-1 is only expressed by lymphatic capillaries (reviewed in8, 9). Moreover, the chemokine CCL21, which is the main attractant of leukocytes expressing the CCR7 chemokine receptor into afferent lymphatics,26 is abundantly expressed in capillaries and displays a mosaic expression pattern in downstream collectors.27, 28 Over the past 15 years, various gene-targeted mouse strains with lymphatic-specific expression of Cre-recombinase (eg, 29-31) or fluorescent proteins have been generated, what has greatly facilitated time-lapse imaging of leukocyte migration into or within afferent lymphatic (Table 1). However, it is important to note that none of the described markers are exclusively expressed in lymphatics; for example, Prox-1 is also expressed in the liver32 and in certain muscle cells,33 LYVE-1 by liver sinusoidal endothelial cells34 and certain macrophages,35 and podoplanin by various stromal cells,36 what may limit the applicability of reporter mice in certain tissue.

TABLE 1. Lymphatic reporter mice and their use to study trafficking through afferent lymphatics Reporter mice First report of mouse line(s) Used to study trafficking through afferent lymphatics Prox1-GFP 119 70, 88 Prox1-mOrange 2 120 88, 115 Prox1-tdTomato 121 No report Prox1-CreERT2 tdTomato 31, 122 122 Prox1-mOrange 2 × CD11c-YFP 120, 123 115 Prox1-GFP × hCD2-DsRed 119, 124 70, 88 VEGFR3-tdTomato 125 No report VE-cadherin-Cre × Rosa-RFP 126, 127 86, 88 Podoplanin GFP-Cre+ 128 No report Lyve1 GFP-Cre+ 129 No report Lyve1-CreERT2-tdTomato 130, 131 No report 4 CELL POPULATIONS MIGRATING THROUGH AFFERENT LYMPHATICS

Cannulation studies, mostly performed 20–50 years ago in larger animals like sheep and also in humans, have revealed that the prevailing cell population in afferent lymph in steady-state conditions is T lymphocytes (80%–90%), specifically antigen-experienced CD4+ effector memory T cells (TEMs).37-40 Contrary, naive T cells, which migrate mainly between blood and SLOs, are found in low numbers in afferent lymph, since they are typically not equipped with homing molecules needed for extravasation into peripheral tissues. DCs, the major antigen-presenting cells, constitute the second most common cell type in afferent lymph (5%–15%). Other cell types such as monocytes and granulocytes or B cells, are present in the lymph but in lower percentages (1%–10%) and rather in the context of inflammatory responses.37, 38, 41, 42 Furthermore, during inflammation, cell migration into afferent lymphatics is substantially increased.39 Interestingly, neutrophils are the first cell type to migrate via afferent lymphatics to dLNs during an inflammatory stimulus.43

Considering the difficulty of cannulating afferent lymphatics in smaller animals, particularly in rodents, only few cannulation experiments performed in mice or rats have been reported.44, 45 Lately, the use of transgenic mice expressing the photoconvertible Kaede or Kikume proteins in all body cells has provided further insights into endogenous lymphatic migration in mice.46, 47 Illumination of a tissue or organ with violet light induces the conversion of cells from a green- to a red-fluorescent state, allowing to identify leukocytes that have emigrated from this site via afferent lymphatics in dLNs by flow cytometry. Interestingly, photoconversion experiments have revealed that under inflammatory conditions the major fraction of T cells emigrating via afferent lymphatics to dLNs are regulatory T cells (Treg).39, 48, 49 However, since Treg where not yet known/analyzed for at the time when most cannulation experiments in sheep or humans were performed, this finding still warrants confirmation in larger animals and humans. Also, considering that laboratory mice comprise a much smaller pool of TEM as compared to feral mice or humans,50 the percentage to Treg migrating in humans is likely to be smaller.

5 PRESUMED SIGNIFICANCE OF MIGRATION THROUGH AFFERENT LYMPHATICS

Although cannulation studies have indicated that DCs are less frequent in afferent lymph than T cells, the importance of their migration has been best studied.26 DCs are professional antigen-presenting cells (APCs) with essential roles in initiation and regulation of adaptive immunity. In peripheral tissues DCs continuously sample their environment and migrate via lymphatics to the first dLN to present peptides derived from ingested proteins on MHC molecules. In absence of an infection, the peptide pool presented will be derived from self-proteins, and DCs will be rather immature, that is, expressing low levels of co-stimulatory molecules. This will prevent potentially self-reactive cognate T cells from being activated but rather induce a state of anergy or tolerance. Consequently, DC migration via afferent lymphatics is considered essential for the maintenance of tolerance. On the other hand, a recent study showed that even in germ-free mice, where most DC migration would be expected to occur under steady-state conditions, T cell activation in dLNs is still observed but suppressed by co-localizing Tregs.51 This illustrates that induction of anergy and tolerance by immature migratory DCs is likely not sufficient for preventing T cell activation across the entire T cell repertoire, highlighting the importance of additional pathways in maintaining peripheral tolerance. Conversely, in the context of infection or inflammation, DCs sense pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), leading to their maturation and enhanced migration to dLNs and effective priming of cognate T cell responses.26 Thus, DC migration from peripheral tissues via afferent lymphatics is considered essential for both induction of adaptive immunity and maintenance of tolerance.

In contrast to DC migration, the functional significance of T cell migration via afferent lymphatics is less clear. During an inflammatory response, for example, caused by an infection, many effector cells (Teff) are typically recruited to the affected tissue.52 Several studies support the notion that retention of Teff at the site of infection occurs in an antigen-specific manner: when encountering a cognate antigen in the context of a viral lung infection, CD4+ Teff have been found to loose residual expression of CCR7, making them less likely to emigrate via CCL21-expressing lymphatics.53 By contrast, unspecific Teff are thought to retain low levels of CCR7, allowing them to sense the chemokine gradient from afferent lymphatics and exit the tissue via this route.54 Similar results were obtained in a delayed-type hypersensitivity (DTH) response induced in murine skin, where CCR7-deficiency in Teff diminished tissue egress and enhanced inflammation.55 Modulation of CCR7 expression in Teff was also shown to impact the strength of the inflammatory response in a murine model of Morbus Crohn: adoptive transfer of CCR7-deficient CD4+ Teff or blockade of CCR7 with antibodies exacerbated the disease.56 Conversely, overexpression of CCR7 increased CD4+ Teff egress from inflamed skin, thereby accelerating resolution of inflammation.56 The regulation of CCR7 expression and tissue exit of unspecific Teff via afferent lymphatic have therefore been brought forward as a mechanism for preventing overshooting tissue inflammation and immune-mediated pathology.

The notion that T cells exiting from inflamed tissues via afferent lymphatics might serve to dampen immune-mediated tissue inflammation is also supported by recent findings regarding Treg migration. As mentioned, in mice Treg have been identified as the major T cell subset exiting inflamed tissues.39, 48, 49 In the case of the skin and colon, it was shown that Treg that have emigrated from these sites to dLNs via afferent LVs display a more suppressive phenotype and exert stronger suppressive activity than Treg that have entered the LN via high endothelial venules (HEVs).39, 48, 49 Similarly, different studies in tissue allograft models showed that Treg migration via afferent lymphatics to dLNs is needed for an efficient downregulation of the adaptive immune response against the allograft.57-59 Interestingly, mice lacking afferent lymphatics were shown to display multiple signs of autoimmunity, what might be a consequence of missing migration of either immature DCs in steady-state or of Treg in inflammation.60 However, considering that—in contrast to mice - the presence and abundance of Treg have thus far not been studied/reported in human afferent lymph, the proof of the potential human relevance of these findings is still missing.

Finally, recirculation of memory T cells through peripheral tissues and their tissue exit via afferent lymphatics is thought to generally contribute to immune surveillance. In addition to tissue-resident memory T cells (TRM) also recirculating memory T cell (TRCM) subsets have recently been identified.61-63 TRCM have been identified in mice62 and humans, where at least two different subsets were discovered.64 As all other skin-exiting T cells, TRCM express CCR7 and use this receptor for migration to dLNs, form where they continue to recirculate via the blood to distal tissues. In contrast, CCR7-/- TRM do not migrate but remain in the tissue where they provide local and immediate protection. Several recent studies have indicated that TRM delay pathogen spread and at the same time serve to “sound the alarm” for the recruitment of TRCM.61 However, in many experimental models of viral or bacterial infection, TRM suffice for immune protection.61, 63 Thus, the exact role of TRCM in immune surveillance and pathogen clearance is not fully understood at present.

6 INSIGHTS INTO LYMPHATIC MIGRATION GAINED FROM MICROSCOPY

Over the past 20 years, in vivo techniques, such as FITC painting, adoptive transfer studies and experiments in photoconvertible mice (discussed in65) have greatly contributed to deciphering the molecular mechanism of lymphatic migration. One downside of these approaches is that they do not allow to dissect the particular anatomic location where a molecule contributes to migration, or how the cellular migration pattern differs at different sites in the tissue or in the vasculature. Contrary to the techniques mentioned above, microscopy allows to study immune cell migration at the single-cell level. Particularly time-lapse imaging approaches have therefore greatly contributed to deciphering the mechanism of cell entry into afferent lymphatics and the intralymphatic behavior of cells, revealing that lymphatic migration occurs in a stepwise manner (Figure 2). The entire process, and in particular the insights gained from imaging, shall be described in greater detail in the following parts.

Lymphatic migration occurs in distinct steps. Leukocytes migrate in a stepwise manner from peripheral tissues via afferent lymphatics to dLNs. (1) Interstitial migration: Leukocytes squeeze and migrate through the interstitial ECM. In vicinity of the lymphatic capillaries, they start to sense the peri-lymphatic CCL21 gradient and begin to directedly migrate toward the vessels. (2) Transmigration into capillaries: Near capillaries, leukocytes squeeze through the thin and discontinuous BM and subsequently transmigrate into the capillary lumen through the open flaps generated between neighboring LECs. (3) Intralymphatic crawling: Once inside the capillaries, leukocytes continue to actively crawl on capillary LECs and patrol the capillary lumen. (4) Passive transport in collectors: Within collecting vessel segments, lymph flow is elevated due to vessel contractions. Once leukocytes have reached this compartment, they start to detach, roll and flow, resulting in rapid transport to the dLN

6.1 Leukocyte entry into lymphatic capillaries

A breakthrough in our understanding of lymphatic migration was achieved in 2008 by the group of Michael Sixt, who introduced a new, nowadays widely used model to image DC entry into lymphatic capillaries in murine ear skin explants66 (Figure 3A). It involves ripping murine ears along the central cartilage to expose the dermal aspects of the dorsal and ventral ear skin, in which lymphatic capillaries can be readily visualized by staining with fluorescent anti-LYVE-1 antibodies. When adding fluorescently labelled DCs onto the explants, DCs rapidly adhere and migrate into the tissue, allowing to visualize the lymphatic entry process by time-lapse imaging. This first report revealed that added DCs rapidly (within 30–60 min) approached and entered into afferent lymphatic capillaries.66 By simultaneously staining for BM components like laminin or collagen IV, the Sixt lab further reported that the capillary-surrounding BM is discontinuous and contains preformed pores through which DCs squeeze during their approach of the lymphatic endothelium.15 Real-time imaging also confirmed that entry into the capillary lumen involved transmigration through the endothelial flaps formed by neighboring oakleaf-shaped LECs,15 in line with what had been hypothesized based on electron microscopy.14

Techniques for time-lapse imaging of leukocyte migration into and within afferent lymphatics. (A) Mouse ear skin explant model (crawl in assay). In this widely used preparation, mouse ears are ripped along the cartilage to expose the dermal tissue aspects. Lymphatics are rapidly stained by fluorescent antibodies (unless using ears of fluorescent lymphatic reporter mice). Subsequently, fluorescent leukocytes (eg, DCs) are added and left to crawl into the tissue. After washing off cells that have not entered the tissue, leukocytes are followed by time-lapse imaging as they approach, enter or crawl within afferent lymphatics. (B) Intravital microscopy (IVM). This image technique allows us to visualize cell dynamics and cell behavior in vivo in anesthetized mice by multiphoton or confocal microscopy. Commonly used sites for imaging are the murine ear skin (depicted), footpad or cremaster muscle. IVM typically requires the use of reporter mice with fluorescent lymphatics and leukocytes, particularly for imaging endogenous leukocytes. Examples shown are from Prox1-GFP × CD2-Dsred mice (green lymphatics, red-fluorescent T cells70) and from Prox1-mOrange2 × CD11c-YFP mice (red lymphatics and yellow DCs,115) 6.2 Integrin requirement of lymphatic migration

Using time-lapse imaging in ear skin explants and adoptive transfer experiments the lab of Michael Sixt further demonstrated that DCs do not require integrins for lymphatic migration in steady-state.66 This finding was rather surprising at the time, considering that leukocyte extravasation from blood vessels is known to be integrin dependent. The reason for this discrepancy likely is that the integrin ligands ICAM-1 and VCAM-1, which also mediate leukocyte extravasation from blood vessels, are much less expressed in LECs than in blood vascular endothelial cells (BECs) in steady-state and are only upregulated under inflammatory conditions.67, 68 Consequently, leukocyte migration through inflamed afferent lymphatics also becomes integrin dependent, as evidence by adoptive transfer and FITC painting experiments.68-70 Besides DCs, the integrin dependency of leukocyte migration from inflamed tissue to dLNs was also confirmed for T cells as well as for neutrophils.70, 71

6.3 CCL21 directs leukocytes toward lymphatic vessels

The dermal ear skin model also generated new insights on how interstitial DCs sense and approach lymphatic capillaries. As previously mentioned, the chemokine CCL21 is constitutively expressed by LECs27, 28, 72 whereas its corresponding chemokine receptor CCR7 is induced in maturing DCs73 and also expressed by the T cell subsets exiting from peripheral tissues. Several studies have identified the CCL21/CCR7 axis as the most important determinant of DC and T cell exit into afferent lymphatics.26, 62, 74

CCL21 contains a highly positively charged C-terminus, which confers binding to glycosaminoglycan-containing heparan sulfates (HS-GAGs) present on the surface of LECs and other cell types.75, 76 However, also other negatively charged molecules, including LEC-expressed podoplanin77 and BM/ECM components have been shown or suggested to bind CCL21.78, 79 Confocal imaging performed in ear skin revealed that most CCL21 is stored intracellularly in the trans-Golgi network.72, 80 Secreted CCL21 gives rise to a peri-lymphatic gradient that decreases toward the interstitium. As revealed by time-lapse imaging in the ear skin model, DCs sense the peri-lymphatic CCL21 gradient starting from approximately 90 μm distance to the vessel wall.72 Surprisingly, a recent study revealed that LEC-expressed HS is not necessary for the formation of the CCL21 gradient, as its loss resulted in an only modest decrease in CCL21 co-localizing with lymphatic capillaries and did not impact DC migration toward capillaries.78 These results suggest that other negatively charged molecules in the LEC glycocalyx or in the LEC-surrounding BM or ECM are the major site of CCL21 anchoring. Adding to the complexity of CCL21 gradient formation, a recent study demonstrated that CCL21 in peripheral tissues exists in a full-length and a more soluble, cleaved version lacking the positively charged C terminus.81

The availability of extracellular CCL21 and the resulting CCL21 gradient appear to be tightly regulated: Under inflammatory conditions, for example, upon stimulation with TNFα, the release of CCL21 from LECs is increased.80 On the other hand, DCs interacting with capillary LECs induce Ca2+ signaling in LECs and trigger the release of intracellular CCL21, thereby depositing a CCL21 track for other migratory DCs.82 Finally, the formation of the extracellular CCL21 gradient is reportedly also regulated by the atypical chemokine receptor 4 (ACKR4), which in the skin is expressed by keratinocytes but also by some fibroblast and LECs.81, 83 ACKR4 scavenges CCL21 as well as CCL19, the second CCR7 ligand, which is upregulated in maturing DCs. In absence of ACKR4, DC migration from skin to dLNs was reduced.81, 83 Moreover, quantitative DC crawl in experiments in murine ear skin explant and intravital imaging in the footpad revealed that in absence of ACKR4 less DCs entered into dermal lymphatic capillaries, likely because of saturation of the immobilized CCL21 gradient.81

6.4 Leukocytes actively crawl within lymphatic capillaries and flow in collectors

In addition to time-lapse imaging in explants, also intravital microscopy (IVM) has provided valuable insights into the cellular and molecular determinants of lymphatic migration (Figure 3B, Table 1). Imaging either in the murine footpad84, 85 or ear skin86 these initial studies confirmed that also in vivo DCs enter into LYVE-1+ lymphatic capillaries. Additionally, they revealed that DCs within lymphatic capillaries were actively crawling at average velocities ranging from 2 to 12 μm/min.84-86 This observation was rather surprising at the time, since in analogy to blood vessels, where leukocytes are typically transported passively with the blood flow, it was initially assumed that leukocytes would immediately detach and flow upon entry into afferent lymphatics. However, it needs to be remembered that the flow in lymphatic capillaries is several orders of magnitude lower than in blood vessels22, 23 likely explaining why leukocytes do not readily flow in lymphatic capillaries. Particularly the work of our group further revealed that DCs in capillaries not only crawled, but rather displayed a patrolling behavior; that is, they frequently turned and also migrated in opposite direction of drainag