Shifting CCR7 towards Its Monomeric Form Augments CCL19 Binding and Uptake.

The chemokine receptor CCR7, together with its ligands, is responsible for the migration and positioning of adaptive immune cells, and hence critical for launching adaptive immune responses. CCR7 is also induced on certain cancer cells and contributes to metastasis formation. Thus, CCR7 expression and signalling must be tightly regulated for proper function. CCR7, like many other members of the G-protein coupled receptor superfamily, can form homodimers and oligomers. Notably, danger signals associated with pathogen encounter promote oligomerisation of CCR7 and is considered as one layer of regulating its function. Here, we assessed the dimerisation of human CCR7 and several single point mutations using split-luciferase complementation assays. We demonstrate that dimerisation-defective CCR7 mutants can be transported to the cell surface and elicit normal chemokine-driven G-protein activation. By contrast, we discovered that CCR7 mutants whose expression are shifted towards monomers significantly augment their capacities to bind and internalise fluorescently labelled CCL19. Modeling of the receptor suggests that dimerisation-defective CCR7 mutants render the extracellular loops more flexible and less structured, such that the chemokine recognition site located in the binding pocket might become more accessible to its ligand. Overall, we provide new insights into how the dimerisation state of CCR7 affects CCL19 binding and receptor trafficking

Distinct Fates of Chemokine and Surrogate Molecule Gradients: Consequences for CCR7-Guided Dendritic Cell Migration.

Chemokine-guided leukocyte migration is a hallmark of the immune system to cope with invading pathogens. Intruder confronted dendritic cells (DCs) induce the expression of the chemokine receptor CCR7, which enables them to sense and migrate along chemokine gradients to home to draining lymph nodes, where they launch an adaptive immune response. Chemokine-mediated DC migration is recapitulated and intensively studied in 3D matrix migration chambers. A major caveat in the field is that chemokine gradient formation and maintenance in such 3D environments is generally not assessed. Instead, fluorescent probes, mostly labelled dextran, are used as surrogate molecules, thereby neglecting important electrochemical properties of the chemokines. Here, we used site-specifically, fluorescently labelled CCL19 and CCL21 to study the establishment and shape of the chemokine gradients over time in the 3D collagen matrix. We demonstrate that CCL19 and particularly CCL21 establish stable, but short-distance spanning gradients with an exponential decay-like shape. By contrast, dextran with its neutral surface charge forms a nearly linear gradient across the entire matrix. We show that the charged C-terminal tail of CCL21, known to interact with extracellular matrix proteins, is determinant for shaping the chemokine gradient. Importantly, DCs sense differences in the shape of CCL19 and CCL21 gradients, resulting in distinct spatial migratory responses

CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70

The chemokine receptor CCR7 guides T cells and dendritic cells to and within lymph nodes to launch the onset of adaptive immunity. Here, we demonstrate that CCR7 in addition acts as a potent co-stimulatory molecule in T cell activation. We found that antigen recognition and engagement of the TCR results in CCR7 accumulation at the immunological synapse where CCR7 and the TCR co-localize within sub-synaptic vesicles. We demonstrate that CCR7 triggering alone is sufficient to recruit and activate ZAP70, a critical kinase for T cell activation, through Src kinase, whereas TCR CCR7 co-stimulation results in increased and prolonged ZAP70 kinase activity. Finally, we show that ZAP70, acting as adapter molecule, is critical for CCR7-mediated inside-out signaling to integrins, thereby modulating LFA-1 valency regulation to promote cell adhesion, a key step in immunological synapse formation and efficient T cell activation

GPR182 is a broadly scavenging atypical chemokine receptor influencing T-independent immunity.

Immune responses highly depend on the effective trafficking of immune cells into and within secondary lymphoid organs (SLOs). Atypical chemokine receptors (ACKRs) scavenge chemokines to eliminate them from the extracellular space, thereby generating gradients that guide leukocytes. In contrast to canonical chemokine receptors, ACKRs do not induce classical intracellular signaling that results in cell migration. Recently, the closest relative of ACKR3, GPR182, has been partially deorphanized as a potential novel ACKR. We confirm and extend previous studies by identifying further ligands that classify GPR182 as a broadly scavenging chemokine receptor. We validate the "atypical" nature of the receptor, wherein canonical G-protein-dependent intracellular signaling is not activated following ligand stimulation. However, β-arrestins are required for ligand-independent internalization and chemokine scavenging whereas the C-terminus is in part dispensable. In the absence of GPR182 in vivo, we observed elevated chemokine levels in the serum but also in SLO interstitium. We also reveal that CXCL13 and CCL28, which do not bind any other ACKR, are bound and efficiently scavenged by GPR182. Moreover, we found a cooperative relationship between GPR182 and ACKR3 in regulating serum CXCL12 levels, and between GPR182 and ACKR4 in controlling CCL20 levels. Furthermore, we unveil a new phenotype in GPR182-KO mice, in which we observed a reduced marginal zone (MZ), both in size and in cellularity, and thus in the T-independent antibody response. Taken together, we and others have unveiled a novel, broadly scavenging chemokine receptor, which we propose should be named ACKR5

CXCL14 preferentially synergizes with homeostatic chemokine receptor systems

Reflecting their importance in immunity, the activity of chemokines is regulated on several levels, including tissue and context-specific expression and availability of their cognate receptor on target cells. Chemokine synergism, affecting both chemokine and chemokine receptor function, has emerged as an additional control mechanism. We previously demonstrated that CXCL14 is a positive allosteric modulator of CXCR4 in its ability to synergize with CXCL12 in diverse cellular responses. Here, we have extended our study to additional homeostatic, as well as a selection of inflammatory chemokine systems. We report that CXCL14 strongly synergizes with low (sub-active) concentrations of CXCL13 and CCL19/CCL21 in in vitro chemotaxis with immune cells expressing the corresponding receptors CXCR5 and CCR7, respectively. CXCL14 by itself was inactive, not only on cells expressing CXCR5 or CCR7 but also on cells expressing any other known conventional or atypical chemokine receptor, as assessed by chemotaxis and/or β-arrestin recruitment assays. Furthermore, synergistic migration responses between CXCL14 and inflammatory chemokines CXCL10/CXCL11 and CCL5, targeting CXCR3 and CCR5, respectively, were marginal and occasional synergistic Ca2+ flux responses were observed. CXCL14 bound to 300-19 cells and interfered with CCL19 binding to CCR7-expressing cells, suggesting that these cellular interactions contributed to the reported CXCL14-mediated synergistic activities. We propose a model whereby tissue-expressed CXCL14 contributes to cell localization under steady-state conditions at sites with prominent expression of homeostatic chemokines.publishe

Heparin Specifically Interacts with Basic BBXB Motifs of the Chemokine CCL21 to Define CCR7 Signaling

Chemokines are critically involved in controlling directed leukocyte migration. Spatiotemporal secretion together with local retention processes establish and maintain local chemokine gradients that guide directional cell migration. Extracellular matrix proteins, particularly glycosaminoglycans (GAGs), locally retain chemokines through electrochemical interactions. The two chemokines CCL19 and CCL21 guide CCR7-expressing leukocytes, such as antigen-bearing dendritic cells and T lymphocytes, to draining lymph nodes to initiate adaptive immune responses. CCL21—in contrast to CCL19—is characterized by a unique extended C-terminus composed of highly charged residues to facilitate interactions with GAGs. Notably, both chemokines can trigger common, but also ligand-biased signaling through the same receptor. The underlying molecular mechanism of ligand-biased CCR7 signaling is poorly understood. Using a series of naturally occurring chemokine variants in combination with newly designed site-specific chemokine mutants, we herein assessed CCR7 signaling, as well as GAG interactions. We demonstrate that the charged chemokine C-terminus does not fully confer CCL21-biased CCR7 signaling. Besides the positively charged C-terminus, CCL21 also possesses specific BBXB motifs comprising basic amino acids. We show that CCL21 variants where individual BBXB motifs are mutated retain their capability to trigger G-protein-dependent CCR7 signaling, but lose their ability to interact with heparin. Moreover, we show that heparin specifically interacts with CCL21, but not with CCL19, and thereby competes with ligand-binding to CCR7 and prevents signaling. Hence, we provide evidence that soluble heparin, but not the other GAGs, complexes with CCL21 to define CCR7 signaling in a ligand-dependent manner

CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70

The chemokine receptor CCR7 guides T cells and dendritic cells to and within lymph nodes to launch the onset of adaptive immunity. Here, we demonstrate that CCR7 in addition acts as a potent co-stimulatory molecule in T cell activation. We found that antigen recognition and engagement of the TCR results in CCR7 accumulation at the immunological synapse where CCR7 and the TCR co-localize within sub-synaptic vesicles. We demonstrate that CCR7 triggering alone is sufficient to recruit and activate ZAP70, a critical kinase for T cell activation, through Src kinase, whereas TCR CCR7 co-stimulation results in increased and prolonged ZAP70 kinase activity. Finally, we show that ZAP70, acting as adapter molecule, is critical for CCR7-mediated inside-out signaling to integrins, thereby modulating LFA-1 valency regulation to promote cell adhesion, a key step in immunological synapse formation and efficient T cell activation.publishe

CCL20 is a novel ligand for the scavenging atypical chemokine receptor 4

The chemokine CCL20 is broadly produced by endothelial cells in the liver, the lung, in lymph nodes and mucosal lymphoid tissues, and recruits CCR6 expressing leukocytes, particularly dendritic cells, mature B cells, and subpopulations of T cells. How CCL20 is systemically scavenged is currently unknown. Here, we identify that fluorescently labeled human and mouse CCL20 are efficiently taken‐up by the atypical chemokine receptor ACKR4. CCL20 shares ACKR4 with the homeostatic chemokines CCL19, CCL21, and CCL25, although with a lower affinity. We demonstrate that all 4 human chemokines recruit β‐arrestin1 and β‐arrestin2 to human ACKR4. Similarly, mouse CCL19, CCL21, and CCL25 equally activate the human receptor. Interestingly, at the same chemokine concentration, mouse CCL20 did not recruit β‐arrestins to human ACKR4. Further cross‐species analysis suggests that human ACKR4 preferentially takes‐up human CCL20, whereas mouse ACKR4 similarly internalizes mouse and human CCL20. Furthermore, we engineered a fluorescently labeled chimeric chemokine consisting of the N‐terminus of mouse CCL25 and the body of mouse CCL19, termed CCL25_19, which interacts with and is taken‐up by human and mouse ACKR4

Naive T lymphocytes chemotax long distance to CCL21 but not to a source of bioactive S1P

International audienceNaive T lymphocytes traffic through the organism in search for antigen, alternating between blood and secondary lymphoid organs. Lymphocyte homing to lymph nodes relies on CCL21 chemokine sensing by CCR7 receptors, while exit into efferent lymphatics relies on sphingolipid S1P sensing by S1PR1 receptors. While both molecules are claimed chemotactic, a quantitative analysis of naive T lymphocyte migration along defined gradients is missing. Here, we used a reductionist approach to study the real-time single-cell response of naive T lymphocytes to CCL21 and serum rich in bioactive S1P. Using microfluidic and micropatterning ad hoc tools, we show that CCL21 triggers stable polarization and long-range chemotaxis of cells, whereas S1P-rich serum triggers a transient polarization only and no significant displacement, potentially representing a brief transmigration step through exit portals. Our in vitro data thus suggest that naive T lymphocyte chemotax long distances to CCL21 but not toward a source of bioactive S1

Genetic Dissection of Aversive Associative Olfactory Learning and Memory in <i>Drosophila</i> Larvae

<div><p>Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult <i>Drosophila</i> it was shown that memory formation after aversive Pavlovian conditioning includes—besides other forms—a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult <i>Drosophila</i>, memory formation was only sporadically analyzed at its larval stage. Here we deconstruct the larval mnemonic organization after aversive olfactory conditioning. We show that after odor-high salt conditioning larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3’5’-monophosphate (cAMP) signaling and <i>synapsin</i> gene function. In addition, we show for the first time for <i>Drosophila</i> larvae an anesthesia resistant component, which relies on <i>radish</i> and <i>bruchpilot</i> gene function, protein kinase C activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution.</p></div