The atypical chemokine receptor CCRL1 shapes functional CCL21 gradients in lymph nodes.

Afferent lymph-borne dendritic cells essentially rely on the chemokine receptor CCR7 for their transition from the subcapsular lymph node sinus into the parenchyma, a migratory step driven by putative gradients of CCR7 ligands. We found that lymph node fringes indeed contained physiological gradients of the chemokine CCL21, which depended on the expression of CCRL1, the atypical receptor for the CCR7 ligands CCL19 and CCL21. Lymphatic endothelial cells lining the ceiling of the subcapsular sinus, but not those lining the floor, expressed CCRL1, which scavenged chemokines from the sinus lumen. This created chemokine gradients across the sinus floor and enabled the emigration of dendritic cells. In vitro live imaging revealed that spatially confined expression of CCRL1 was necessary and sufficient for the creation of functional chemokine gradients.

A 4-midable Connection: CCR7 Tetramers Link GPCR to Src Kinase Signaling.

Chemokine receptors are known to signal through heterotrimeric G proteins. In this issue, Hauser et al. (2016) report that inflammatory cues can induce tetramers of the chemokine receptor CCR7 that serve as scaffolds integrating G protein with Src kinase signaling.

Afferent lymph-derived T cells and DCs use different chemokine receptor CCR7-dependent routes for entry into the lymph node and intranodal migration.

Little is known about the molecular mechanisms that determine the entry into the lymph node and intranodal positioning of lymph-derived cells. By injecting cells directly into afferent lymph vessels of popliteal lymph nodes, we demonstrate that lymph-derived T cells entered lymph-node parenchyma mainly from peripheral medullary sinuses, whereas dendritic cells (DCs) transmigrated through the floor of the subcapsular sinus on the afferent side. Transmigrating DCs induced local changes that allowed the concomitant entry of T cells at these sites. Signals mediated by the chemokine receptor CCR7 were absolutely required for the directional migration of both DCs and T cells into the T cell zone but were dispensable for the parenchymal entry of lymph-derived T cells and dendrite probing of DCs. Our findings provide insight into the molecular and structural requirements for the entry into lymph nodes and intranodal migration of lymph-derived cells of the immune system.

Lymph node homing of T cells and dendritic cells via afferent lymphatics.

The continuous migration of immune cells is of utmost importance for the induction of both protective immunity as well as immunological tolerance. However, relatively little is known about the molecular cues that regulate the entry of immune cells from peripheral, nonlymphoid tissues into afferent lymph vessels and, in particular, their subsequent transmigration from afferent lymphatics into the parenchyma of draining lymph nodes (LNs). Here, we review the requirements for T cells and dendritic cells (DCs) to enter initial afferent lymph vessels of the skin. We discuss how these cells subsequently gain access to the paracortex of draining lymph nodes; a location that allows for efficient interaction between both cell populations, providing the right environment for the induction of immunity as well as tolerance.

CCR7-mediated LFA-1 functions in T cells are regulated by 2 independent ADAP/SKAP55 modules.

The ß2-integrin lymphocyte function-associated antigen-1 (LFA-1) plays a crucial role within the immune system. It regulates the interaction between T cells and antigen-presenting cells and facilitates T-cell adhesion to the endothelium, a process that is important for lymphocyte extravasation and homing. Signals mediated via the T-cell receptor and the chemokine receptor CCR7 activate LFA-1 through processes known as inside-out signaling. The molecular mechanisms underlying inside-out signaling are not completely understood. Here, we have assessed the role of the ADAP/SKAP55 module for CCR7-mediated signaling. We show that loss of the module delays homing and reduces intranodal T-cell motility in vivo. This is probably because of a defect in CCR7-mediated adhesion that affects both affinity and avidity regulation of LFA-1. Further analysis of how the ADAP/SKAP55 module regulates CCR7-induced integrin activation revealed that 2 independent pools of the module are expressed in T cells. One pool interacts with a RAPL/Mst1 complex, whereas the other pool is linked to a RIAM/Mst1/Kindlin-3 complex. Importantly, both the RAPL/Mst1 and the RIAM/Mst1/Kindlin-3 complexes require ADAP/SKAP55 for binding to LFA-1 upon CCR7 stimulation. Hence, 2 independent ADAP/SKAP55 modules are essential components of the signaling machinery that regulates affinity and avidity of LFA-1 in response to CCR7.

Rapid leukocyte migration by integrin-independent flowing and squeezing.

All metazoan cells carry transmembrane receptors of the integrin family, which couple the contractile force of the actomyosin cytoskeleton to the extracellular environment. In agreement with this principle, rapidly migrating leukocytes use integrin-mediated adhesion when moving over two-dimensional surfaces. As migration on two-dimensional substrates naturally overemphasizes the role of adhesion, the contribution of integrins during three-dimensional movement of leukocytes within tissues has remained controversial. We studied the interplay between adhesive, contractile and protrusive forces during interstitial leukocyte chemotaxis in vivo and in vitro. We ablated all integrin heterodimers from murine leukocytes, and show here that functional integrins do not contribute to migration in three-dimensional environments. Instead, these cells migrate by the sole force of actin-network expansion, which promotes protrusive flowing of the leading edge. Myosin II-dependent contraction is only required on passage through narrow gaps, where a squeezing contraction of the trailing edge propels the rigid nucleus.

CCR7 ligands stimulate the intranodal motility of T lymphocytes in vivo.

In contrast to lymphocyte homing, little is known about molecular cues controlling the motility of lymphocytes within lymphoid organs. Applying intravital two-photon microscopy, we demonstrate that chemokine receptor CCR7 signaling enhances the intranodal motility of CD4(+) T cells. Compared to wild-type (WT) cells, the average velocity and mean motility coefficient of adoptively transferred CCR7-deficient CD4(+) T lymphocytes in T cell areas of WT recipients were reduced by 33 and 55%, respectively. Both parameters were comparably reduced for WT T lymphocytes migrating in T cell areas of plt/plt mice lacking CCR7 ligands. Importantly, systemic application of the CCR7 ligand CCL21 was sufficient to rescue the motility of WT T lymphocytes inside T cell areas of plt/plt recipients. Comparing the movement behavior of T cells in subcapsular areas that are devoid of detectable amounts of CCR7 ligands even in WT mice, we failed to reveal any differences between WT and plt/plt recipients. Furthermore, in both WT and plt/plt recipients, highly motile T cells rapidly accumulated in the subcapsular region after subcutaneous injection of the CCR7 ligand CCL19. Collectively, these data identify CCR7 and its ligands as important chemokinetic factors stimulating the basal motility of CD4(+) T cells inside lymph nodes in vivo.

CCR7 essentially contributes to the homing of plasmacytoid dendritic cells to lymph nodes under steady-state as well as inflammatory conditions.

The chemokine receptor CCR7 represents an important determinant for circulating lymphocytes to enter lymph nodes (LN) via high endothelial venules. High endothelial venules also represent the major site of entry for plasmacytoid dendritic cells (pDC). In the steady-state, murine pDC have been suggested to home to LN engaging the chemokine receptors CXCR3, CXCR4, and CCR5, whereas responsiveness to CCR7 ligands is thought to be acquired only upon activation. In this study, we show that already resting pDC express minute amounts of CCR7 that suffice to trigger migration to CCL19/CCL21 in vitro. Upon activation with TLR ligands, CCR7 levels on pDC are strongly increased. Notably, CCR7-deficient mice display substantially reduced pDC counts in LN but not in bone marrow and spleen. Adoptive cell transfer experiments revealed that under both steady-state as well as inflammatory conditions, the homing of CCR7-deficient pDC is severely impaired, indicating that the reduced cell counts of naive pDC observed in CCR7(-/-) mice reflect an intrinsic homing defect of pDC. Together, these observations provide strong evidence that similar to naive lymphocytes, nonstimulated pDC exploit CCR7 to gain entry into LN. This adds to the repertoire of chemokine receptors permitting them to enter diverse tissues.

Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells.

Oral tolerance induction is a key feature of intestinal immunity, generating systemic nonresponsiveness to ingested antigens. In this study, we report that orally applied soluble antigens are exclusively recognized in the intestinal immune system, particularly in the mesenteric lymph nodes. Consequently, the initiation of oral tolerance is impeded by mesenteric lymphadenectomy. Small bowel transplantation reveals that mesenteric lymph nodes require afferent lymph to accomplish the recognition of orally applied antigens. Finally, oral tolerance cannot be induced in CCR7-deficient mice that display impaired migration of dendritic cells from the intestine to the mesenteric lymph nodes, suggesting that immunologically relevant antigen is transported in a cell-bound fashion. These results demonstrate that antigen transport via afferent lymphatics into the draining mesenteric lymph nodes is obligatory for oral tolerance induction, inspiring new therapeutic strategies to exploit oral tolerance induction for the prevention and treatment of autoimmune diseases.

Tolerance induction towards cardiac allografts under costimulation blockade is impaired in CCR7-deficient animals but can be restored by adoptive transfer of syngeneic plasmacytoid dendritic cells.

Deficiency of transplant recipients for the chemokine receptor CCR7 was originally described to slightly increase the survival time of vascularized solid organ grafts, probably due to a reduced priming of alloreactive T cells. Using a model of allotolerance induction by donor-specific splenocyte transfusion (DST) in combination with anti-CD40L mAb-mediated costimulation blockade (CSB), we show here a striking failure of CCR7-deficient (CCR7(-/-) ) recipients to tolerate cardiac allografts. Furthermore, in addition to the recently described lack of Treg, CCR7(-/-) mice were found to harbor significantly reduced numbers of plasmacytoid dendritic cells (pDCs) within peripheral as well as mesenteric lymph nodes (LNs), but not the bone marrow or spleen. pDCs had previously been suggested to function as tolerogenic APC during allograft transplantation, and a single transfer of syngeneic WT pDCs, but not conventional DCs, was indeed sufficient to rescue graft survival in DST+CSB-treated CCR7(-/-) recipients in a dose-dependent manner. We therefore conclude that the nearly complete absence of pDCs within LNs of CCR7(-/-) mice prevents the successful induction of DST+CSB-mediated allotolerance, leading to the observed acute rejection of cardiac allografts under tolerizing conditions.

T cell-dendritic cell interaction dynamics during the induction of respiratory tolerance and immunity.

Dendritic cells (DCs) residing in the lung are known to acquire inhaled Ag and, after migration to the draining bronchial lymph node (brLN), to present it to naive T cells in an either tolerogenic or immunogenic context. To visualize endogenous lung-derived DCs, we applied fluorescent latex beads (LXs) intratracheally, thereby in vivo labeling the majority of phagocytic cells within the lung. Of note, LX-bearing cells subsequently arriving in the draining brLN were found to represent lung-derived migratory DCs. Imaging explanted brLN by two-photon laser-scanning microscopy, we quantitatively analyzed the migration and interaction behavior of naive CD4(+) T cells and endogenous, lung-derived DC presenting airway-delivered Ag under inflammatory or noninflammatory conditions. Ag-specific naive CD4(+) T cells engaged in stable as well as transient contacts with LX-bearing DCs in both situations and displayed similar overall motility kinetics, including a pronounced decrease in motility at 16-20 h after antigenic challenge. In contrast, the comparative analysis of T cell-DC cluster sizes as well as contact durations strongly suggests that lung-derived migratory DCs and naive CD4(+) T cells form more stable, long-lasting contacts under inflammatory conditions favoring the induction of respiratory immunity.

Intra- and intercompartmental movement of gammadelta T cells: intestinal intraepithelial and peripheral gammadelta T cells represent exclusive nonoverlapping populations with distinct migration characteristics.

Unlike the ∼1% of γδ TCR-positive T cells being regularly present in blood and secondary lymphoid organs (peripheral γδ T cells), ∼50-60% of small intestinal intraepithelial lymphocytes (iIELs) in the mouse express the γδ TCR (γδ iIELs). In this study, we investigated the overlap and exchange of γδ iIELs and γδ T cells found in peripheral secondary lymphoid organs. Using two-photon laser-scanning microscopy, we found γδ T cells within peripheral lymph nodes to be highly motile, whereas γδ iIELs were characterized by a locally confined scanning behavior. Our results implied a strict separation of peripheral γδ T cells and γδ iIELs. Nevertheless, γδ iIELs could be efficiently regenerated from bone marrow-derived precursors in irradiated or T cell-deficient adult mice. However, outside the intestinal epithelium, survival of γδ iIELs was very poor. In CCR9-deficient mice, homing of γδ iIELs was impaired, but did not lead to an accumulation of γδ iIEL-like cells in the periphery. Conversely, in situations in which specific γδ iIEL niches were empty, adoptive transfer of isolated γδ iIELs led to a sustained engraftment of transferred γδ iIELs in the intestinal epithelium for at least 100 d. Furthermore, we demonstrated by heterotopic intestinal transplantation experiments that an exchange of γδ iIELs only rarely happens in the steady state of adult mice. We therefore conclude that peripheral versus intestinal intraepithelial γδ T cells are exclusive, nonoverlapping populations that virtually do not exchange with each other.

Factors governing the intranodal migration behavior of T lymphocytes.

With the advent of two-photon microscopic imaging, the last few years have witnessed remarkable progress regarding our understanding of the movement behavior and interaction dynamics of different immune cells within lymphoid organs. The basal intranodal motility of naive T lymphocytes in the absence of antigenic or inflammatory stimuli, although at first glance representing a phenomenon of apparent simplicity, is far from being completely understood. The most important open question in this context relates to the origin of intranodal T-cell motility itself: how are these highly dynamic cells 'motivated' to carry out their relentless scanning of dendritic cells present in the lymph node (LN)? This review summarizes recent advances in the search for factors governing the intranodal migration behavior of naive T lymphocytes, focusing in particular on the role of extracellular pro-migratory cues, intracellular signaling components, and the influence of the structural LN environment on intranodal T-cell motility.

Constant TCR triggering suggests that the TCR expressed on intestinal intraepithelial γδ T cells is functional in vivo.

Intestinal intraepithelial lymphocytes carrying the γδ TCR (γδ iIEL) are involved in the maintenance of epithelial integrity. γδ iIEL have an activated phenotype, characterized by CD69 expression and increased cell size compared with systemic T lymphocytes. As an additional activation marker, the majority of γδ iIEL express the CD8αα homodimer. However, our knowledge about cognate ligands for most γδ TCR remains fragmentary and recent advances show that γδ T cells including iIEL may be directly activated by cytokines or through NK-receptors, TLR and other pattern recognition receptors. We therefore asked whether the TCR of γδ iIEL was functional beyond its role during thymic selection. Using TcrdH2BeGFP (Tcrd, T-cell receptor δ locus; H2B, histone 2B) reporter mice to identify γδ T cells, we measured their intracellular free calcium concentration in response to TCR-crosslinking. In contrast to systemic γδ T cells, CD8αα(+) γδ iIEL showed high basal calcium levels and were refractory to TCR-dependent calcium-flux induction; however, they readily produced CC chemokine ligand 4 (CCL4) and IFN-γ upon TCR triggering in vitro. Notably, in vivo blocking of the γδ TCR with specific mAb led to a decrease of basal calcium levels in CD8αα(+) γδ iIEL. This suggests that the γδ TCR of CD8αα(+) γδ iIEL is constantly being triggered and therefore functional in vivo.

T cell migration dynamics within lymph nodes during steady state: an overview of extracellular and intracellular factors influencing the basal intranodal T cell motility.

Naive T lymphocytes continuously recirculate through secondary lymphoid organs such as lymph nodes until they are eventually activated by recognizing cognate peptide/MHC-complexes on the surface of antigen-protecting cells. The intranodal T cell migration behavior leading to these crucial--and potentially rare--encounters during the induction of an adaptive immune response could not be directly addressed until, in 2002, the use of two-photon microscopy also allowed the visualization of cellular dynamics deep within intact lymph nodes. Since then, numerous studies have confirmed that, by default, naive T cells are extremely motile, scanning the paracortical T cell zone for cognate antigen by means of an apparent random walk. This review attempts to summarize the current knowledge of factors influencing the basal migration behavior of naive T lymphocytes within lymph nodes during steady state. Extracellular cues, such as the motility-promoting influence of CCR7 ligands and the role of integrins during interstitial migration, as well as intracellular signaling pathways involved in T cell motility, will be discussed. Particular emphasis is placed on structural features of the lymph node environment orchestrating T cell migration, namely the framework of fibroblastic reticular cells serving as migration "highways." Finally, new approaches to simulate the cellular dynamics within lymph nodes in silico by means of mathematical modeling will be reviewed.

Efficient homing of T cells via afferent lymphatics requires mechanical arrest and integrin-supported chemokine guidance.

Little is known regarding lymph node (LN)-homing of immune cells via afferent lymphatics. Here, we show, using a photo-convertible Dendra-2 reporter, that recently activated CD4 T cells enter downstream LNs via afferent lymphatics at high frequencies. Intra-lymphatic immune cell transfer and live imaging data further show that activated T cells come to an instantaneous arrest mediated passively by the mechanical 3D-sieve barrier of the LN subcapsular sinus (SCS). Arrested T cells subsequently migrate randomly on the sinus floor independent of both chemokines and integrins. However, chemokine receptors are imperative for guiding cells out of the SCS, and for their subsequent directional translocation towards the T cell zone. By contrast, integrins are dispensable for LN homing, yet still contribute by increasing the dwell time within the SCS and by potentially enhancing T cell sensing of chemokine gradients. Together, these findings provide fundamental insights into mechanisms that control homing of lymph-derived immune cells.

Dendritic cell migration in health and disease.

Dendritic cells (DCs) are potent and versatile antigen-presenting cells, and their ability to migrate is key for the initiation of protective pro-inflammatory as well as tolerogenic immune responses. Recent comprehensive studies have highlighted the importance of DC migration in the maintenance of immune surveillance and tissue homeostasis, and also in the pathogenesis of a range of diseases. In this Review, we summarize the anatomical, cellular and molecular factors that regulate the migration of different DC subsets in health and disease. In particular, we focus on new insights concerning the role of migratory DCs in the pathogenesis of diseases of the skin, intestine, lung, and brain, as well as in autoimmunity and atherosclerosis.

Interleukin-23-Dependent γ/δ T Cells Produce Interleukin-17 and Accumulate in the Enthesis, Aortic Valve, and Ciliary Body in Mice.

OBJECTIVE: The spondyloarthritides (SpA) are a group of rheumatic diseases characterized by ossification and inflammation of entheseal tissue, the region where tendon attaches to bone. Interleukin-23 (IL-23) is involved in the pathogenesis of SpA by acting on IL-23 receptor (IL-23R) expressed on enthesis-resident lymphocytes. Upon IL-23 binding, CD3+CD4-CD8- tissue-resident lymphocytes secrete IL-17A and IL-22, leading to inflammation, bone loss, and ossification. Knowledge about enthesis-resident lymphocytes remains fragmentary, and the contribution of entheseal γ/δ T cells in particular is not clear. This study was undertaken to investigate the presence of γ/δ T cells in the enthesis. METHODS: We used 2-photon microscopy and flow cytometry to analyze entheseal lymphocytes from C57BL/6, Tcrd-H2BeGFP, Rorc-GFP, and IL-23R-eGFP mice. To analyze entheseal γ/δ T cells in IL-23-induced inflammation, Tcrd-H2BeGFP mice were crossed with mice of the susceptible B10.RIII background. Hydrodynamic injection of IL-23 minicircle DNA was performed for overexpression of IL-23 and induction of inflammation. Light-sheet fluorescence microscopy was used to visualize arthritic inflammation. RESULTS: Activated Vγ6+CD27- γ/δ T cells were abundant in uninflamed entheseal tissue and constituted the large majority of retinoic acid receptor-related orphan nuclear receptor γt (RORγt)+IL-23R+ enthesis-resident lymphocytes. Fetal thymus-dependent γ/δ T cells were the main source of IL-17A at the enthesis. Under inflammatory conditions, γ/δ T cells increased in number at the Achilles tendon enthesis, aortic root, and adjacent to the ciliary body. CONCLUSION: Entheseal γ/δ T cells are derived from fetal thymus and are maintained as self-renewing tissue-resident cells. As main IL-17A producers within tissues exposed to mechanical stress including enthesis, γ/δ T cells are key players in the pathogenesis of IL-23-induced local inflammation.

Helicobacter hepaticus induces an inflammatory response in primary human hepatocytes.

Helicobacter hepaticus can lead to chronic hepatitis and hepatocellular carcinoma in certain strains of mice. Until now the pathogenic role of Helicobacter species on human liver tissue is still not clarified though Helicobacter species identification in human liver cancer was successful in case controlled studies. Therefore we established an in vitro model to investigate the interaction of primary human hepatocytes (PHH) with Helicobacter hepaticus. Successful co-culturing of PHH with Helicobacter hepaticus was confirmed by visualization of motile bacteria by two-photon-microscopy. Isolated human monocytes were stimulated with PHH conditioned media. Changes in mRNA expression of acute phase cytokines and proteins in PHH and stimulated monocytes were determined by Real-time PCR. Furthermore, cytokines and proteins were analyzed in PHH culture supernatants by ELISA. Co-cultivation with Helicobacter hepaticus induced mRNA expression of Interleukin-1 beta (IL-1ß), Tumor necrosis factor-alpha, Interleukin-8 (IL-8) and Monocyte chemotactic protein-1 (MCP-1) in PHH (p<0.05) resulting in a corresponding increase of IL-8 and MCP-1 concentrations in PHH supernatants (p<0.05). IL-8 and IL-1ß mRNA expression was induced in monocytes stimulated with Helicobacter hepaticus infected PHH conditioned media (p<0.05). An increase of Cyclooxygenase-2 mRNA expression was observed, with a concomitant increase of prostaglandin E2 concentration in PHH supernatants at 24 and 48 h (p<0.05). In contrast, at day 7 of co-culture, no persistent elevation of cytokine mRNA could be detected. High expression of intercellular adhesion molecule-1 on PHH cell membranes after co-culture was shown by two-photon-microscopy and confirmed by flow-cytometry. Finally, expression of Cytochrome P450 3A4 and albumin mRNA were downregulated, indicating an impairment of hepatocyte synthesis function by Helicobacter hepaticus presence. This is the first in vitro model demonstrating a pathogenic effect of a Helicobacter spp. on human liver cells, resulting in an inflammatory response with increased synthesis of inflammatory mediators and consecutive monocyte activation.

Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions.

Chemokine receptor CX3CR1(+) dendritic cells (DCs) have been suggested to sample intestinal antigens by extending transepithelial dendrites into the gut lumen. Other studies identified CD103(+) DCs in the mucosa, which, through their ability to synthesize retinoic acid (RA), appear to be capable of generating typical signatures of intestinal adaptive immune responses. We report that CD103 and CX3CR1 phenotypically and functionally characterize distinct subsets of lamina propria cells. In contrast to CD103(+) DC, CX3CR1(+) cells represent a nonmigratory gut-resident population with slow turnover rates and poor responses to FLT-3L and granulocyte/macrophage colony-stimulating factor. Direct visualization of cells in lymph vessels and flow cytometry of mouse intestinal lymph revealed that CD103(+) DCs, but not CX3CR1-expressing cells, migrate into the gut draining mesenteric lymph nodes (LNs) under steady-state and inflammatory conditions. Moreover, CX3CR1(+) cells displayed poor T cell stimulatory capacity in vitro and in vivo after direct injection of cells into intestinal lymphatics and appeared to be less efficient at generating RA compared with CD103(+) DC. These findings indicate that selectively CD103(+) DCs serve classical DC functions and initiate adaptive immune responses in local LNs, whereas CX3CR1(+) populations might modulate immune responses directly in the mucosa and serve as first line barrier against invading enteropathogens.